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

Gene name - polo

Synonyms -

Cytological map position - 77A3--77A3

Function - mitotic protein kinase

Keywords - cell cycle, centriole

Symbol - polo

FlyBase ID:FBgn0003124

Genetic map position - 3-46.

Classification - protein kinase, CDC5 homolog

Cellular location - cytoplasmic, associated with chromosomes during mitosis

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Ramdas Nair, A., Singh, P., Salvador Garcia, D., Rodriguez-Crespo, D., Egger, B. and Cabernard, C. (2016). The microcephaly-associated protein Wdr62/CG7337 is required to maintain centrosome asymmetry in Drosophila neuroblasts. Cell Rep [Epub ahead of print]. PubMed ID: 26804909
Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates, but the underlying molecular mechanisms are incompletely understood. This study reports that loss of CG7337, the fly ortholog of WDR62, compromises interphase centrosome asymmetry in fly neural stem cells (neuroblasts). Wdr62 maintains an active interphase microtubule-organizing center (MTOC) by stabilizing microtubules (MTs), which are necessary for sustained recruitment of Polo/Plk1 to the pericentriolar matrix (PCM) and downregulation of Pericentrin-like protein (Plp). The loss of an active MTOC in wdr62 mutants compromises centrosome positioning, spindle orientation, and biased centrosome segregation. wdr62 mutant flies also have an approximately 40% reduction in brain size as a result of cell-cycle delays. It is proposed that CG7337/Wdr62, a microtubule-associated protein, is required for the maintenance of interphase microtubules, thereby regulating centrosomal Polo and Plp levels. Independent of this function, Wdr62 is also required for the timely mitotic entry of neural stem cells.

Eisman, R. C., Phelps, M. A. and Kaufman, T. C. (2016). The End of a Monolith: Deconstructing the Cnn-Polo interaction. Fly (Austin): [Epub ahead of print]. PubMed ID: 27096551
In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. A recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at two residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the two phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.

Zhu, K., Shan, Z., Zhang, L. and Wen, W. (2016). Phospho-Pon binding-mediated fine-tuning of Plk1 activity. Structure [Epub ahead of print]. PubMed ID: 27238966
In Drosophila neuroblasts (NBs), the asymmetrical localization and segregation of the cell-fate determinant Numb are regulated by its adaptor Partner of Numb (Pon) and the cell-cycle kinase Polo. Polo phosphorylates the Pon localization domain, thus leading to its basal distribution together with Numb, albeit through an unclear mechanism. This study finds that Cdk1 phosphorylates Pon at Thr63, thus creating a docking site for the Polo-box domain (PBD) of Polo-like kinase 1 (Plk1). The crystal structure of the Plk1 PBD/phospho-Pon complex reveals that two phospho-Pon bound PBDs associate to form a dimer of dimers. Evidence is provided that phospho-Pon binding-induced PBD dimerization relieves the autoinhibition of Plk1. Moreover, the priming Cdk1 phosphorylation of Pon was shown to be important for sequential Plk1 phosphorylation. These results not only provide structural insight into how phosphoprotein binding activates Plk1 but also suggest that binding to different phosphoproteins might mediate the fine-tuning of Plk1 activity.

Pimenta-Marques, A., Bento, I., Lopes, C.A., Duarte, P., Jana, S.C. and Bettencourt-Dias, M. (2016). A mechanism for the elimination of the female gamete centrosome in Drosophila melanogaster. Science [Epub ahead of print]. PubMed ID: 27229142
An important feature of fertilization is the asymmetric inheritance of centrioles. In most species it is the sperm that contributes the initial centriole, which builds the first centrosome that is essential for early development. However, given that centrioles are thought to be exceptionally stable structures, the mechanism behind centriole disappearance in the female germline remains elusive and paradoxical. Using fruit flies, this study elucidated a program for centriole maintenance. This program is led by Polo kinase and the pericentriolar matrix (PCM). The PCM is down-regulated in the female germline during oogenesis, which results in centriole loss. Perturbing this program prevents centriole loss, leads to abnormal meiotic and mitotic divisions, and thus to female sterility. This mechanism challenges the view that centrioles are intrinsically stable structures and reveals general functions for Polo kinase and the PCM in centriole maintenance. The study propose that regulation of this maintenance program is essential for successful sexual reproduction, and defines centriole life span in different tissues in homeostasis and disease, shaping the cytoskeleton.

Novak, Z. A., Wainman, A., Gartenmann, L. and Raff, J. W. (2016). Cdk1 phosphorylates Drosophila Sas-4 to recruit Polo to daughter centrioles and convert them to centrosomes. Dev Cell 37: 545-557. PubMed ID: 27326932
Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic 'centriole conversion' is regulated, but it requires Plk1/Polo kinase. This study shows that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome.
Esmaeeli-Nieh, S., et al. (2016). BOD1 is required for cognitive function in humans and Drosophila. PLoS Genet 12: e1006022. PubMed ID: 27166630
This study reports a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 (see Drosophila Polo) substrates by modulating Protein Phosphatase 2A (PP2A; see Drosophila Twins) activity during mitosis. Fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and evidence is provided for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, two Drosophila models were established, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together these results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila evidence is provided for a conserved role of BOD1 in the development and maintenance of cognitive features.
Brose, L., Crest, J., Tao, L. and Sullivan, W. (2017). Polo kinase mediates the phosphorylation and cellular localization of Nuf/FIP3, a Rab11 effector. Mol Biol Cell [Epub ahead of print]. PubMed ID: 28381422
Animal cytokinesis involves both actin-myosin based contraction and vesicle-mediated membrane addition. In many cell types, including early Drosophila embryos, Nuf/FIP3, a Rab11 effector, mediates recycling endosome (RE)-based vesicle delivery to the cytokinesis furrow. Nuf exhibits a cell cycle-regulated concentration at the centrosome that is accompanied by dramatic changes in its phosphorylation state. This study demonstrates maximal phosphorylation of Nuf occurs at prophase, when centrosome-associated Nuf disperses throughout the cytoplasm. Accordingly, ectopic Cdk1 activation results in immediate Nuf dispersal from the centrosome. Screening of candidate kinases reveals a specific, dosage-sensitive interaction between Nuf and Polo with respect to Nuf-mediated furrow formation. Inhibiting Polo activity results in Nuf under-phosphorylation and prolonged centrosome association. In vitro, Polo directly binds and is required for Nuf phosphorylation at Ser225 and Thr227, matching previous in vivo mapped phosphorylation sites. These results demonstrate a role for Polo kinase in directly mediating Nuf cell cycle-dependent localization.
Li, Z., Cui, Q., Xu, J., Cheng, D., Wang, X., Li, B., Lee, J. M., Xia, Q., Kusakabe, T. and Zhao, P. (2017). SUMOylation regulates the localization and activity of Polo-like kinase 1 during cell cycle in the silkworm, Bombyx mori. Sci Rep 7(1): 15536. PubMed ID: 29138491
Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator by its specific localization and activity during cell cycle. It has been shown that the phosphorylation and ubiquitylation of Plk1 are required for its own activation and localization. This study reports that SUMOylation regulates the activity of Plk1 in the lepidopteran insect of Bombyx mori. In the absence of SUMOylation, it causes the lost localization of Plk1 on centrosomes and kinetochores, as well as an uneven distribution in midzone. It was further identified that the putative SUMOylation site of Bombyx Plk1 at lysine 466 is required for its localization on centrosomes, and K466 mutation in Plk1 could influence its interaction with Smt3/Ubc9 complex. These findings are also confirmed by Drosophila Polo and human Plk1, which together reveals a conserved role of Plk1 SUMOylation in mammals. Moreover, conjugation of Smt3 to Plk1 SUMOylation mutant promotes its localization on centrosomes and kinetochores, and rescues functional defects of chromosome alignment in cells depleted of endogenous Plk1. Altogether, the present data indicate that the SUMOylation of Plk1 could participate in proper chromosome alignment and segregation during mitosis, and provides a novel layer for the regulation of Plk1 localization and activity throughout cell cycle.
Kachaner, D., Garrido, D., Mehsen, H., Normandin, K., Lavoie, H. and Archambault, V. (2017). Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry. Nat Commun 8(1): 1701. PubMed ID: 29167465
The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. Polo is composed of an N-term kinase domain (KD) and a C-term polo-box domain (PBD), which regulates its subcellular localizations. The PBD and KD can interact and inhibit each other, and this reciprocal inhibition is relieved when Polo is phosphorylated at its activation loop. How Polo activation and localization are coupled during mitotic entry is unknown. This study reports that PBD binding to the KD masks a nuclear localization signal (NLS). Activating phosphorylation of the KD leads to exposure of the NLS and entry of Polo into the nucleus before nuclear envelope breakdown. Failures of this mechanism result in misregulation of the Cdk1-activating Cdc25 phosphatase and lead to mitotic and developmental defects in Drosophila. These results uncover spatiotemporal mechanisms linking master regulatory enzymes during mitotic entry.
Bonner, A. M. and Hawley, R. S. (2018). Functional consequences of the evolution of matrimony, a meiosis-specific inhibitor of Polo kinase. Mol Biol Evol. PubMed ID: 30351378
Meiosis is a defining characteristic of eukaryotes, believed to have evolved only once, over one billion years ago. While the general progression of meiotic events is conserved across multiple diverse organisms, the specific pathways and proteins involved can be highly divergent, even within species from the same genus. This study investigate the rapid evolution of Matrimony (Mtrm), a female meiosis-specific regulator of Polo kinase (Polo) in Drosophila. Mtrm physically interacts with Polo and is required to restrict the activity of Polo during meiosis. Despite Mtrm's critical role in meiosis, sequence conservation within the genus Drosophila is poor. To explore the functional significance of this rapid divergence, Mtrm proteins from 12 different Drosophila species were expressed in the D. melanogaster female germline. Distantly related Mtrm homologs are able to both physically interact with D. melanogaster Polo and rescue the meiotic defects seen in mtrm mutants. However, these distant homologs are not properly degraded after the completion of meiosis. Rather, they continue to inhibit Polo function in the early embryo, resulting in dominant maternal-effect lethality. The ability of Mtrm to be properly degraded, and thus release Polo, is partially due to residues or motifs found within Mtrm's least-conserved regions. It is hypothesized that, while Mtrm regions critical for its meiotic function are under strong purifying selection, changes that occurred in its unconserved regions may have been advantageous, potentially by affecting the timing or duration of meiosis and/or the early embryonic divisions.
Ramani, A., Mariappan, A., Gottardo, M., Mandad, S., Urlaub, H., Avidor-Reiss, T., Riparbelli, M., Callaini, G., Debec, A., Feederle, R. and Gopalakrishnan, J. (2018). Plk1/Polo phosphorylates Sas-4 at the onset of mitosis for an efficient recruitment of pericentriolar material to centrosomes. Cell Rep 25(13): 3618-3630.e3616. PubMed ID: 30590037
Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. This study identified that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4 localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and gamma-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and gamma-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.


Mitosis is a highly regulated process that assures the proper allotment of genetic material between each pair of daughter cells. It proceeds through successive stages of well defined and coordinated sub-processes. Entry into mitosis is regulated by the cdc2/Cyclin B heterodimer. Cdc2/Cyclin B activity drives the events of early mitosis, such as nuclear breakdown, chromosome condensation and spindle formation by phosphorylating cellular substrates. While cdc2 (the catalytic subunit of the heterodimer) is required to drive the events of early mitosis, it must then be inactivated to allow the events of late mitosis to proceed.

Polo is an evolutionarily conserved kinase, active during mitosis. In Drosophila the Polo kinase activity peaks from between late anaphase to telophase, later than the peak of cdc2-Cyclin B kinase activity (which is highest in cells just entering mitosis). polo mutants show an accumulation of cells with condensed chromosomes. Although the extent of chromosome condensation is more like that normally seen in metaphase, the distribution is more typical of prophase; the chromosomes are not aligned at the metaphase plate. Thus it appears that chromosome condensation continues to occur even though other aspects of mitosis, such as alignment at the metaphase plate are delayed (Llamazares, 1991).

polo also exhibits a meiotic phenotype. Meiotic spindles found in mutant testes are generally irregular in shape and structure. The irregular shape of the Nebenkern (mitochondria) in mutants indicates an unequal partitioning of mitochondria on the meiotic spindles, and ultimately into daughter cells. Non-disjunction (separation) of chromosomes during meiosis, is also apparent (Sunkel, 1988).

How does Polo kinase affect mitosis? One must look to research in other organisms for an answer to this question. In Xenopus a Polo related kinase (Plx1) targets Cdc25 (Drosophila homolog: String), the phosphatase that dephosphorylates and consequently activates the cyclin dependent kinase cdc2. Xenopus Plx1 undergoes activation and phosphorylation by multiple kinases at mitosis; it is a kinase that associates with and phosphorylates the amino-terminal domain of Cdc25. It is likely that Plx1 participates in the control of mitotic progression by targeting Cdc25 (Kumagai, 1996).

Studies in yeast suggest additional roles for a Polo-like kinase. The budding yeast gene CDC5 and S. pombe gene plo1 are homologous to polo. Loss of plo1 function results in mitotic arrest; condensed chromosomes are associated with a monopolar spindle, suggesting a failure in formation of a normal bipolar spindle. In addition, mutation can also result in the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of a filamentous actin ring, and in the deposition of septal material, suggesting that PLO1 protein is required high in the regulatory cascade that controls septation. Overexpression can also induce septum formation in G2 cells. It therefore appears that PLO1 is involved in a cascade that leads to bipolar spindle formation, and in a separately regulated chain of events that results in actin ring formation and septum deposition, both of which are required for septum formation (Ohkura, 1995).

Yeast plo1 shows a genetic interaction with cut7 (Ohkura, 1995), closely related to Drosophila kinesin-like protein KLP61F that is essential for mitosis. In the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. KLP61F is important for spindle pole separation and mitotic spindle dynamics. Does Polo target kinesins in higher eukaryotes? (Heck, 1994)

A Polo mammalian homolog has been shown to target a Kinesin protein involved in the cross-bridging of antiparallel microtubules during mitosis. Murine PLK is a Polo like kinase that accumulates after serum stimulation of tissue culture cells. In cells that are continuously cycling between growth and mitosis, Plk protein begins to accumulate at the DNA synthetic/G2 phase boundary and reaches a maximum level at the G2/Mitosis boundary. Plk enzymatic activity gradually decreases as mitosis proceeds, but persists longer than cyclin B-associated cdc2 kinase activity, a similar expression pattern to that found with Drosophila Polo. At the interzone in anaphase, Plk is localized to the area surrounding the spindle axis; during telophase and cytokinesis, it finally concentrates at the midbody. Plk and Mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bindling and antiparallel movement in vitro, are colocalized during late M phase. In addition, MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk kinase activity in vitro. It thus appears that Plk targets a kinesin protein involved in the structuring of microtubules (Lee, 1995).

It is not yet clear whether Polo acts independently of cyclin dependent kinase activity or acts downstream of cyclin dependent kinase. Yeast CDC5 appears to be cdc2 dependent (Ohkura, 1995), while murine Plk activity is not directly regulated by cdc2 or MAP kinase. Neither kinase is able to phosphorylate or regulate the activity of Plk (Lee, 1995). In either case, Polo kinase provides an excellent example of the complexity of regulation that accompanies mitosis. The conservation of Polo and its function in organisms as diverse as yeast and mammals suggests that the cascade of events that regulate mitosis are evolutionarily conserved. Glover (1996) provides an excellent review or the role of Polo in mitosis.

PP2A-twins is antagonized by greatwall and collaborates with polo for cell cycle progression and centrosome attachment to nuclei in drosophila embryos

Cell division and development are regulated by networks of kinases and phosphatases. In early Drosophila embryogenesis, 13 rapid nuclear divisions take place in a syncytium, requiring fine coordination between cell cycle regulators. The Polo kinase is a conserved, crucial regulator of M-phase. An antagonism exists between Polo and Greatwall (Gwl), another mitotic kinase, in Drosophila embryos (Archambault, 2007). However, the nature of the pathways linking them remained elusive. A comprehensive screen was conducted for additional genes functioning with polo and gwl. A strong interdependence was uncovered between Polo and Protein Phosphatase 2A (PP2A) with its B-type subunit Twins (Tws). Reducing the maternal contribution of Polo and PP2A-Tws together is embryonic lethal. Polo and PP2A-Tws were found to collaborate to ensure centrosome attachment to nuclei. While a reduction in Polo activity leads to centrosome detachments observable mostly around prophase, a reduction in PP2A-Tws activity leads to centrosome detachments at mitotic exit, and a reduction in both Polo and PP2A-Tws enhances the frequency of detachments at all stages. Moreover, Gwl was shown to antagonize PP2A-Tws function in both meiosis and mitosis. This study highlights how proper coordination of mitotic entry and exit is required during embryonic cell cycles and defines important roles for Polo and the Gwl-PP2A-Tws pathway in this process (Wang, 2011).

These results shed new light on cell cycle regulation and syncytial embryogenesis. High Polo activity is needed to promote the normal cohesion between centrosomes and nuclei, and this is mostly observable around the time of mitotic entry. Interestingly, transiently detached centrosomes can be recaptured by the assembling spindle and nuclear division can then be completed. This centrosome recapture is probably essential for successful development of the syncytial embryo. A systematic genetic screen unveiled a very strong and specific functional link between Polo and a specific form of PP2A associated with its B-type subunit Tws. PP2A-Tws activity is required for centrosome cohesion with nuclei, although in late M-phase, around the time of mitotic exit. This is consistent with a recent study where centrosome defects were observed in late M-phase when the small T antigen of SV40, which binds PP2A, was expressed in Drosophila embryos (Kotadia, 2008}. PP2A-B55δ (ortholog of Twins) has been recently implicated in promoting mitotic exit in vertebrates, by inactivating Cdc25C and by directly dephosphorylating Cdk1 mitotic substrates (Castilho, 2009; Forester, 2007). The closely related isoform PP2A-B55α has been shown to promote the timely reassembly of the nuclear envelope at mitotic exit. Thus, the failure to reattach centrosomes to nuclei during mitotic exit in PP2A-Tws compromised embryos could be due to problems or a delay in nuclear envelope resealing (Wang, 2011).

The results indicate that the proper regulation of the events of mitotic entry and exit by Polo and PP2A-Tws is crucial. This may be particularly true in the syncytial embryo due to the rapidity of the cycles, where one mitosis is almost immediately followed by another, and because of the obligatory cohesion between centrosomes and nuclei for their migration towards the cortex of the syncytium. Combining partial decreases in the activities of Polo and Tws strongly enhances the frequency of centrosome detachments observed. This suggests that when centrosomes fail to attach properly for too long between mitotic exit and the next mitotic entry, they become permanently detached from nuclei, leading to failures in mitotic divisions (Wang, 2011).

The differences in timing between the detachments observed in polo and tws hypomorphic situations led to a proposal that the two enzymes act in parallel pathways, of which the disruption can lead to a failure in centrosome-nucleus cohesion. This is also supported by the prominent roles of Polo in regulating centrosome maturation and mitotic entry (Archambault, 2009), and the specific requirements of PP2A-Tws/B55 at mitotic exit. However, it cannot be excluded that Polo, Gwl and PP2A-Tws could function on a common substrate, or even in the same linear pathway, where the different players of the pathway could become more or less influential at different times of the cell cycle. In has been proposed that PP2A promotes full expression of Polo in larval neuroblasts and in S2 cells (Wang, 2009). It has also been shown that depletion of Tws by RNAi leads to centrosome maturation defects in S2 cells (Dobbelaere, 2008), which could be explained by a reduction in Polo levels. However, no significant difference has been detected in Polo levels in embryos from gwlScant/+ or tws/+ females, compared to wild-type controls by Western blotting. Deeper genetic and molecular dissection of those pathways should lead to a clearer understanding of the regulation of centrosome and nuclear dynamics during mitotic entry and exit (Wang, 2011).

These results add strong support to an emerging model for a pathway that controls entry into and exit from mitosis and meiosis in animal cells. It is increasingly clear that a form of PP2A associated with a B-type regulatory subunit plays a crucial and conserved role in competing with Cdk1. In Xenopus egg extract, PP2A-B55δ activity is high in interphase and low in M phase. PP2A-B55δ must be down-regulated to allow mitotic entry, and conversely, it appears to promote mitotic exit both by inactivating Cdc25C and by dephosphorylating Cdk1 substrates. In human cells, depletion in B55α delays the events of mitotic exit, including nuclear envelope reassembly. Already some years ago, mutations in Drosophila tws were found to lead to a mitotic arrest in larval neuroblasts, and extracts from tws mutants were shown to have a reduced ability to dephosphorylate Cdk substrates. Mutations in mts resulted in an accumulation of nuclei in mitosis in the embryo. The budding yeast now appears to be a particular case, as its strong reliance on the Cdc14 phosphatase to antagonize Cdk1 may reflect the need for insertion of the anaphase spindle through the bud neck prior to mitotic exit, a constraint that does not exist in animal cells. Nevertheless, additional phosphatases to PP2A, including PP1 are likely to play conserved roles in promoting mitotic and meiotic exit, and this remains to be dissected (Wang, 2011 and references therein).

Identification of PP2A genes as functional interactors of polo and gwl is the result of an unbiased genetic screen. It was found that an elevation in Gwl function combined with a reduction in PP2A-Tws activity leads to a block in M phase, either in metaphase of meiosis I or in the early mitotic cycles. However, positioning of Gwl as an antagonist of PP2A-Tws was facilitated by reports that appeared subsequent to the screen, proposing that the main role of Gwl in promoting M-phase was to lead to the inactivation of PP2A-B55δ in Xenopus egg extracts. Results consistent with this idea were also obtained in mammalian cells (Wang, 2011 and references therein).

More recently, two seminal biochemical studies using Xenopus egg extracts showed that the antagonism of PP2A-B55δ by Gwl is mediated by α-endosulfine/Ensa and Arpp19, two small, related proteins which, when phosphorylated by Gwl at a conserved serine residue, become able to bind and inhibit PP2A-B55δ (Gharbi-Ayachi, 2010; Mochida, 2010). By this mechanism, Gwl activation at mitotic entry leads to the inhibition of PP2A-B55γ, which results in an accumulation of the phosphorylated forms of Cdk1 substrates. Depletion of human Arpp19 also perturbs mitotic progression in Hela cells (Gharbi-Ayachi, 2010), suggesting a conserved role among vertebrates (Wang, 2011).

In an independent study, the group of David Glover has recently identified mutations in Drosophila endosulfine (endos) as potent suppressors of the embryonic lethality that occurs when gwlScant (the gain-of-function allele) is combined with a reduction in polo function, in a maternal effect (Rangone, 2011). endos is the single fly ortholog of Xenopus α-endosulfine and Arpp19. That the identification of endos by Rangone came from another unbiased genetic screen testifies of the specificity and conservation of the Gwl-Endos-PP2A pathway in animal cells. The authors went as far as showing that the critical phosphorylation site of Gwl in Endos is conserved between frogs and flies, and is critical for the function of Endos in antagonizing PP2A-Tws in cultured cells. These findings are consistent with a previous report showing that mutations in endos lead to a failure of oocytes to progress into meiosis until metaphase I (Von Stetina, 2008). Moreover, loss of Gwl specifically in the female germline also leads to meiotic failure, although in that case oocytes do reach metaphase I but exit the arrest aberrantly (Archambault, 2007). Although the meaning of those phenotypic differences is not yet understood, Gwl and Endos are both required for meiotic progression in Drosophila. Conversely, this study shows that excessive Gwl activity relative to PP2A-Tws prevents exit from the metaphase I arrest, suggesting that the inhibition of PP2A-Tws by Gwl and Endos must be relieved to allow completion of meiosis. Moreover, Rangone (2011) shows that the Endos pathway also regulates the mitotic cell cycle in the early embryo, in larval neuroblasts and in cultured cells (Wang, 2011).

Together, the systematic and unbiased identifications of mutations in PP2A-Tws subunit genes as enhancers (this paper), and of mutations in endos as suppressors (Rangone, 2011) of gwlScant provide strong evidence for a pathway connecting these genes to control M phase in flies. These studies provide a convincing genetic and functional validation of the recent biochemical results from Xenopus extracts, and show that the Gwl-Endos-PP2A-Tws/B55 pathway is conserved and plays a key role in regulating both meiosis and mitosis in a living animal (Wang, 2011).

POLO ensures chromosome bi-orientation by preventing and correcting erroneous chromosome-spindle attachments

Correct chromosome segregation during cell division requires bi-orientation at the mitotic spindle. Cells possess mechanisms to prevent and correct inappropriate chromosome attachment. Sister kinetochores assume a 'back-to-back' geometry on chromosomes that favors amphitelic orientation but the regulation of this process and molecular components are unknown. Abnormal chromosome-spindle interactions do occur but are corrected through the activity of Aurora B, which destabilizes erroneous attachments. This study addresses the role of Drosophila POLO in chromosome-spindle interactions and shows that, unlike inhibition of its activity, depletion of the protein results in bipolar spindles with most chromosomes forming stable attachments with both sister kinetochores bound to microtubules from the same pole in a syntelic orientation. This is partly the result of impaired localization and activity of Aurora B but also of an altered centromere organization with abnormal distribution of centromeric proteins and shorter interkinetochore distances. These results suggests that POLO is required to promote amphitelic attachment and chromosome bi-orientation by regulating both the activity of the correction mechanism and the architecture of the centromere (Moutinho-Santos, 2012).

Chromosome bi-orientation relies upon a correction mechanism, which actively promotes the destabilization of chromosome attachment errors, and a prevention mechanism, which acts to reduce inaccurate chromosome orientations. The current findings show that POLO kinase is a major regulator of proper chromosome attachments and bi-orientation by intervening in both mechanisms. On the one hand, this study found that POLO is essential for the centromeric localization and activity of Aurora B, the key kinase in the correction mechanism, so that without POLO destabilization of improper attachments does not take place. A recent study (Foley, 2011) also showed that both Plk1 and Aurora B kinases have a role in the destabilization of the of kinetochore attachment to the microtubule that is counter-balanced by the centromeric localization and activity of the B56-PP2A phosphatase. In contrast, this study has observed that depletion of POLO causes the displacement of proteins within the centromere and considerable shortening of interkinetochore distances, indicating that this kinase is essential for the architecture of the centromere and, therefore, the spatial organization of sister kinetochores. In addition, it is also well established that POLO-like kinases regulate resolution of chromosome arms, the decatenation enzyme topoisomerase II and the condensin II complex. Therefore, it is plausible that alteration of the chromosomal topology caused by lack of POLO function could affect centromeric architecture (Moutinho-Santos, 2012).

The observation that POLO is necessary for proper chromosome attachment because it participates directly in both error-prevention and error-correction mechanisms has important implications for how bi-orientation is achieved, and sheds new light on the extent to which the geometry of the kinetochore pair contributes to the accuracy of chromosome attachments. The current results suggest a major contribution of the prevention mechanism in avoiding attachment errors in metazoan cells (Moutinho-Santos, 2012).

A Meiosis-Specific Form of the APC/C Promotes the Oocyte-to-Embryo Transition by Decreasing Levels of the Polo Kinase Inhibitor Matrimony

Oocytes are stockpiled with proteins and mRNA that are required to drive the initial mitotic divisions of embryogenesis. But are there proteins specific to meiosis whose levels must be decreased to begin embryogenesis properly? The Drosophila protein Cortex (Cort) is a female, meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. Immunoprecipitation of Cortex followed by mass spectrometry was performed, and the Polo kinase inhibitor Matrimony (Mtrm) was identified as a potential interactor with Cort. In vitro binding assays showed Mtrm and Cort can bind directly. Mtrm protein levels are reduced dramatically during the oocyte-to-embryo transition, and this downregulation does not take place in cort mutant eggs, consistent with Mtrm being a substrate of APCCort. Mtrm was shown to be subject to APCCort-mediated proteasomal degradation, and a putative APC/C recognition motif was identified in Mtrm that when mutated partially stabilized the protein in the embryo. Furthermore, overexpression of Mtrm in the early embryo caused aberrant nuclear divisions and developmental defects, and these were enhanced by decreasing levels of active Polo. These data indicate APC(Cort) ubiquitylates Mtrm at the oocyte-to-embryo transition, thus preventing excessive inhibition of Polo kinase activity due to Mtrm's presence (Whitfield, 2013).

Despite its pivotal role in development, regulation of the oocyte-to-embryo transition is poorly understood. Given the maternal stockpiles in the oocyte, mechanistic differences between meiosis and mitosis, and meiosis-specific forms of the APC/C, it is crucial to determine which proteins need to be degraded to switch correctly from meiosis to mitosis. The meiosis-specific activator Cort is essential for the transition from oocyte to embryo despite Fzy/Cdc20's presence. Cortex's existence raised the possibility that degradation of particular meiosis-specific proteins may be necessary for the onset of embryogenesis. This study shows this to be the case: the Cort form of the APC/C is required for Mtrm's destruction at the oocyte-to-embryo transition. Furthermore, reduced levels of Mtrm heading into embryogenesis are necessary for proper development, indicative of requirements for differential levels of the protein in meiosis and mitosis (Whitfield, 2013).

A requirement for reduction in levels of Mtrm is illustrated by the deleterious effects of overexpression of the protein in the embryo. A crucial role for Mtrm degradation in the transition from oocyte to embryo is supported by the observation that reduction in levels of Mtrm protein can suppress the developmental block caused by low activity of Cort. In the grau mutants, levels of Cort are reduced, and the mutant oocytes arrest in meiosis. By mutating a single copy of the mtrm gene, this arrest was overcome, the eggs progressed, and several nuclear divisions occurred (Whitfield, 2013).

Mtrm provides key insights into how protein degradation can be regulated at the oocyte-to-embryo transition. Mtrm is not completely removed from the embryo, illustrating that its protein levels are important and degradation does not have to be an all-or-none process. In this case, APCCort acts as a rheostat, allowing for high levels of Mtrm in meiosis and low levels in mitosis. Consistent with this, it is interesting that stabilized forms of Mtrm present at lower levels than the overexpressed wild-type form did not exhibit an embryonic phenotype. mCherry-Mtrm also is present at levels lower than endogenous Mtrm in stage 14 oocytes, and therefore may never reach high enough levels to be able to cause the developmental defects seen with the overexpressed form of Mtrm. This offers evidence for a specific threshold of Mtrm that can be tolerated in the early embryo (Whitfield, 2013).

Polo kinase is a critical regulator of both mitosis and meiosis, and is conserved from yeast to humans. polo (and its orthologs) help regulate mitotic/meiotic entry, chromosome segregation, centrosome dynamics, and cytokinesis. With such diverse roles during mitosis and meiosis, Polo function must be carefully regulated. Up-regulation of human Polo-like kinase (Plk1) is prevalent in many human cancers, and identifying potent inhibitors of Plk1 is the focus of much research. In Drosophila, without inhibition by Mtrm during prophase of meiosis I, Polo prematurely triggers nuclear envelope breakdown (through activation of the Cdc25 phosphatase) and eventually leads to chromosome nondisjunction. Mutation of polo has direct consequences on female meiotic progression as well. During Drosophila embryogenesis, expression of Scant, a hyperactive form of the Polo antagonist Greatwall kinase, leads to dissociated centrosomes from prophase nuclei. Embryos homozygous for polo1 show a wide array of defects, including irregular DNA masses with disorganized spindles, reminiscent of the mtrm overexpression phenotype. These data illustrate the importance of Polo kinase in both mitosis and meiosis, and that improper regulation of its activity can have disastrous consequences on cell division (Whitfield, 2013).

Current evidence suggests that Mtrm regulates Polo activity during both meiosis and mitosis. The current results shed light on how the oocyte/embryo might use the same protein to regulate Polo during such drastically different cell divisions. The data indicate meiosis requires high levels of Mtrm protein/Polo inhibition, while low levels of Mtrm are needed for early embryogenesis. This is likely a mechanism to allow for fine tuning of Polo activity during the rapid divisions of the syncytial embryo (Whitfield, 2013).

The results of this study provide an interesting biological counterpoint to a recent study on the S. cerevisiae meiosis-specific APC/C activator Ama1. Previously, Ama1 had been known to act later in meiosis, regulating spore formation and Cdc20 degradation at meiosis II, It has been shown that APCAma1 also acts earlier in meiosis to clear out mitotic regulators (including Polo/Cdc5) during the extended meiotic prophase I. Consequently, cells lacking Ama1 exit prematurely from prophase I. It is interesting that two meiosis-specific APC/C activators have now been tied to regulation of Polo kinase. Ama1 has a direct, inhibitory effect early in meiosis, whereas Cort seemingly activates Polo indirectly through degradation of Mtrm late in meiosis (Whitfield, 2013).

Mtrm is not likely to be the only specific substrate of Cort, and it will be exciting to search for more APCCort substrates in the future. It will also be interesting to examine whether Cort targets continue to follow a graded versus all-or-none pattern of degradation during the oocyte-to-embryo transition. Further study of meiosis-specific APC/C activators will give valuable insight into the distinctions between meiotic and mitotic regulation and the control of the onset of embryogenesis (Whitfield, 2013).


Transcript size - 2.2 and 2.5 kb

cDNA clone length - 2221 and 2541

Bases in 5' UTR - 219

Exons - 5

Bases in 3' UTR - 330 and 593


Amino Acids - 577

Structural Domains

The amino terminal 277 amino aids show considerable identity with catalytic domains of protein kinases (Llamazares, 1991).

polo: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 April 2007 

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