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 link: Entrez Gene
polo orthologs: Biolitmine
Recent literature
Wang, L. I., Das, A. and McKim, K. S. (2019). Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments. PLoS Genet 15(5): e1008072. PubMed ID: 31150390
Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. This study found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. This study has demonstrated that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, this study proposes that two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.
Bonner, A. M., Hughes, S. E. and Hawley, R. S. (2020). Regulation of Polo Kinase by Matrimony Is Required for Cohesin Maintenance during Drosophila melanogaster Female Meiosis. Curr Biol. PubMed ID: 32008903
The Drosophila PLK Polo kinase (Polo) is inhibited by the female meiosis-specific protein Matrimony (Mtrm) in a stoichiometric manner. Drosophila Polo localizes strongly to kinetochores and to central spindle microtubules during prometaphase and metaphase I of female meiosis. Mtrm protein levels increase dramatically after nuclear envelope breakdown. This study shows that Mtrm is enriched along the meiotic spindle and that loss of mtrm results in mislocalization of the catalytically active form of Polo. The mtrm gene is haploinsufficient, and heterozygosity for mtrm results in high levels of achiasmate chromosome missegregation. In mtrm/(+) heterozygotes, there is a low level of sister centromere separation, as well as precocious loss of cohesion along the arms of achiasmate chromosomes. However, mtrm-null females are sterile, and sister chromatid cohesion is abolished on all chromosomes, leading to a failure to properly congress or orient chromosomes in metaphase I. These data demonstrate a requirement for the inhibition of Polo, perhaps by sequestering Polo to the microtubules during Drosophila melanogaster female meiosis and suggest that catalytically active Polo is a distinct subset of the total Polo population within the oocyte that requires its own regulation.
Landmann, C., Pierre-Elies, P., Goutte-Gattat, D., Montembault, E., Claverie, M. C. and Royou, A. (2020). The Mre11-Rad50-Nbs1 complex mediates the robust recruitment of Polo to DNA lesions during mitosis. J Cell Sci. PubMed ID: 32487663
The DNA damage sensor, Mre11-Rad50-Nbs1 complex, and Polo kinase are recruited to DNA lesions during mitosis. However, their mechanism of recruitment is elusive. Using live-cell imaging combined with the micro-irradiation of single chromosomes, this study analyzed the dynamics of Polo and Mre11 at DNA lesions during mitosis. The two proteins display distinct kinetics. While Polo kinetics at DSBs are Cdk1-driven, Mre11 promptly but briefly associates with DSBs regardless of the phase of mitosis and re-associates with DSBs in the proceeding interphase. Mechanistically, Polo kinase activity is required for its own recruitment and that of the mitotic proteins BubR1 and Bub3 to DSBs. Moreover, depletion of Rad50 severely impaired Polo kinetics at mitotic DSBs. Conversely, ectopic tethering of Mre11 to chromatin is sufficient to recruit Polo. This study highlights a novel pathway that links the DSB sensor MRN complex and Polo kinase to initiate a prompt, decisive response to the presence of DNA damage during mitosis.
Morgunova, V., Kordyukova, M., Mikhaleva, E. A., Butenko, I., Pobeguts, O. V. and Kalmykova, A. (2021). Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development. PLoS One 16(10): e0258156. PubMed ID: 34624021
Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. The mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila was studied. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. This study found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction (Morgunona, 2021).
Gallaud, E., Richard-Parpaillon, L., Bataille, L., Pascal, A., Metivier, M., Archambault, V. and Giet, R. (2022). The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation. PLoS Genet 18(4): e1010145. PubMed ID: 35377889
The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Using asymmetrically dividing Drosophila larval neural stem cells (NSCs) as a model, it was shown that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, it was shown that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, these results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.
Yang, S., McAdow, J., Du, Y., Trigg, J., Taghert, P. H. and Johnson, A. N. (2022). Spatiotemporal expression of regulatory kinases directs the transition from mitosis to cellular morphogenesis in Drosophila. Nat Commun 13(1): 772. PubMed ID: 35140224
Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. This study shows the mitotic kinase Polo, which regulates all steps of mitosis in Drosophila, also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis. In the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases.
Deng, Q., Wang, C., Koe, C. T., Heinen, J. P., Tan, Y. S., Li, S., Gonzalez, C., Sung, W. K. and Wang, H. (2022). Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells. PLoS Biol 20(10): e3001834. PubMed ID: 36223339
Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. This study demonstrated that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, human Parafibromin was shown to rescue the ectopic NSC phenotype in Drosophila hyx mutant brains. This study also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. These data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers.
Wong, S. S., Wilmott, Z. M., Saurya, S., Alvarez-Rodrigo, I., Zhou, F. Y., Chau, K. Y., Goriely, A. and Raff, J. W. (2022). Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly. Embo j 41(11): e110891. PubMed ID: 35505659
Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. This study shows that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process-peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. It is proposed that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.
Hayden, L., Hur, W., Vergassola, M. and Di Talia, S. (2022). Manipulating the nature of embryonic mitotic waves. Curr Biol 32(22): 4989-4996.e4983. PubMed ID: 36332617
Early embryogenesis is characterized by rapid and synchronous cleavage divisions, which are often controlled by wave-like patterns of Cdk1 activity. Two mechanisms have been proposed for mitotic waves: sweep and trigger waves. The two mechanisms give rise to different wave speeds, dependencies on physical and molecular parameters, and spatial profiles of Cdk1 activity: upward sweeping gradients versus traveling wavefronts. Both mechanisms hinge on the transient bistability governing the cell cycle and are differentiated by the speed of the cell-cycle progression: sweep and trigger waves arise for rapid and slow drives, respectively. This study, using quantitative imaging of Cdk1 activity and theory, illustrates that sweep waves are the dominant mechanism in Drosophila embryos and test two fundamental predictions on the transition from sweep to trigger waves. Sweep waves can be turned into trigger waves if the cell cycle is slowed down genetically or if significant delays in the cell-cycle progression are introduced across the embryo by altering nuclear density. Genetic experiments demonstrate that Polo kinase is a major rate-limiting regulator of the blastoderm divisions, and genetic perturbations reducing its activity can induce the transition from sweep to trigger waves. Furthermore, it was shown that changes in temperature cause an essentially uniform slowdown of interphase and mitosis. That results in sweep waves being observed across a wide temperature range despite the cell-cycle durations being significantly different. Collectively, the combination of theory and experiments elucidates the nature of mitotic waves in Drosophila embryogenesis, their control mechanisms, and their mutual transitions.
Loh, M., Bernard, F. and Guichet, A. (2023). Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte. Development 150(13). PubMed ID: 37334771
Microtubules and their associated motors are important players in nucleus positioning. Although nuclear migration in Drosophila oocytes is controlled by microtubules, a precise role for microtubule-associated molecular motors in nuclear migration has yet to be reported. This study characterized novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, it is reported that, before migration, the nucleus moves from the oocyte anterior side toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In the absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. The maintenance of a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning. In the absence of Kinesin-1, SPD-2, an essential component of the pericentriolar material, is increased at the centrosomes, suggesting that Kinesin-1-associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. Our results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity.


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).

The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation

Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis - a process termed centrosome maturation. How centrosomes mature remains mysterious. This study identified a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. It is concluded that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies (Conduit, 2014).

As cells enter mitosis, centrosomes mature, and the amount of PCM recruited around the centrioles dramatically increases. Although many proteins have been implicated in this process, little is known about how they organize a functional mitotic centrosome. Previous studies have hinted at the existence of a PCM scaffold, but its molecular nature has remained elusive. The current data suggest that Cnn is phosphorylated specifically at centrosomes during mitosis, and this phosphorylation allows Cnn to assemble into a scaffold around the centrioles. Perturbing Cnn phosphorylation prevents efficient scaffold assembly and efficient mitotic PCM recruitment, demonstrating that the phosphorylated Cnn scaffold plays an important part in centrosome maturation in flies (Conduit, 2014).

This study demonstrates unambiguously that the Cnn scaffold is in constant flux: as the Cnn scaffold spreads slowly outward, it is continuously replenished by new phosphorylated Cnn that assembles around the centrioles; in this way, the Cnn scaffold is built from the inside out. This inside-out assembly mechanism has important implications, because it potentially explains how centrioles can influence the size of the PCM and organize centrosomes of different sizes within the same cell - as seems to occur in several asymmetrically dividing stem/progenitor cells (Conduit, 2014).

How does Cnn assemble into a scaffold structure? Cnn contains a PReM domain that contains a LZ and ten Ser/Thr residues that are highly conserved in Drosophila species. Mutating the LZ or the ten Ser/Thr residues to Ala strongly inhibits Cnn scaffold assembly in vivo, while mutating these ten Ser/Thr residues to phosphomimicking residues promotes spontaneous Cnn scaffold assembly in the cytosol, independently of centrosomes. Moreover, whereas the WT PReM domain predominantly forms dimers via the LZ in vitro, replacing the ten Ser/Thr residues with phosphomimicking residues allows the PReM domain to assemble into higher-order multimers in an LZ-dependent manner. Modeling suggests that the arrangement of hydrophobic and hydrophilic residues within the LZ could allow multiple LZs to associate laterally to form such multimeric structures. It is speculated, therefore, that these stable multimers formed by the phosphomimicking mutant PReM domains in vitro may be the fundamental building blocks of the phosphorylated Cnn scaffold in vivo. How these multimers assemble into a larger macromolecular scaffold is unclear, but Y2H analysis indicates that multiple regions of Cnn can self-interact and so could potentially participate in such a process (Conduit, 2014).

How is Cnn scaffold assembly regulated so that it only occurs during mitosis? Polo/Plk1 is a key regulator of PCM assembly in many systems and it is activated in human cells during the G2/M transition. In flies, knocking down Polo in cultured fly cells abolishes Cnn phosphorylation and strongly perturbs Cnn's centrosomal localization. This study shows that recombinant human Plk1 can phosphorylate the PReM domain of Cnn in vitro and that at least six of the putative phosphorylation sites within the PReM domain conform to a Polo/Plk1 recognition motif. Moreover, abolishing these putative phosphorylation sites prevents Cnn phosphorylation in vitro and Cnn scaffold formation in vivo, whereas mutating these sites to phosphomimicking residues promotes multimerization in vitro and spontaneous scaffold formation in vivo. Thus, it seems likely that Polo is activated during mitosis in fly cells and directly phosphorylates Cnn to initiate Cnn scaffold assembly, although the possibility cannot be excluded that Polo activates an unknown kinase that then phosphorylates Cnn (Conduit, 2014).

How is Cnn scaffold assembly regulated so that it only occurs around the centrioles? The data strongly indicate that Cnn is normally phosphorylated exclusively at centrosomes, and Polo is highly concentrated at centrioles throughout the cell cycle. While it remains formally possible that Cnn is phosphorylated in the cytosol and phosphorylated Cnn is then rapidly sequestered at centrosomes, this is thought unlikely for two reasons: (1) phosphomimetic Cnn is not rapidly transported to centrosomes, but rather spontaneously assembles into scaffolds in the cytoplasm, and (2) in mitotic extracts of brain cells that lack centrosomes, phosphorylated Cnn cannot be detected. It is interesting that the phosphorylation of at least six of the ten conserved Ser/Thr residues within thePReMdomain appears to be required for efficient scaffold assembly. The potential advantages of regulation by multisite phosphorylation in allowing switch-like transitions are well documented. Thus, it seems likely that the requirement for multisite phosphorylation helps ensure that Cnn normally only efficiently forms a scaffold around the centrioles, where there is a high concentration of both the kinase and its substrate. Cnn is a large protein that contains several predicted coiledcoil regions, supporting the idea that it can act as a molecular scaffold onto which other PCM proteins can assemble. Proteins related to Cnn have been identified in species ranging from yeasts to humans, and many of these proteins have been implicated in centrosome or MT organizing center assembly; they are also usually large proteins with several predicted coiled-coil domains, and some family members have been shown to interact directly with several other PCM components, including the γlTuRC, Aurora A, and Pericentrin. Althoug no obvious PReM domain has been identified in vertebrate Cnn family members, many of these proteins have regions that might fulfill the minimal requirements for a PReMlike domain - a potential coiled-coil interaction domain, and a region containing multiple potential phosphorylation sites. It is therefore suspected that Cnn-like proteins will contribute to PCM scaffold formation in many systems (Conduit, 2014).

Evidence that a positive feedback loop drives centrosome maturation in fly embryos

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. This study shows that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. These findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).

Centrosomes play an important part in many aspects of cell organisation, and they form when a mother centriole recruits pericentriolar material (PCM) around itself. The PCM contains several hundred proteins, allowing the centrosome to function as a major microtubule (MT) organising centre, and also as an important coordination centre and signalling hub. Centrosome dysfunction has been linked to several human diseases and developmental disorders, including cancer, microcephaly and dwarfism (Alvarez-Rodrigo, 2019).

During interphase, the mother centriole recruits a small amount of PCM that is highly organised. As cells prepare to enter mitosis, however, the PCM expands dramatically around the mother centriole in a process termed centrosome maturation. Electron microscopy (EM) studies suggest that centrioles organise an extensive 'scaffold' structure during mitosis that surrounds the mother centriole and recruits other PCM components such as the γ-tubulin ring complex (γ-TuRC) (Alvarez-Rodrigo, 2019).

In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, a relatively simple pathway seems to govern the assembly of this mitotic PCM scaffold. The conserved centriole/centrosome protein Spd-2/SPD-2 (fly/worm nomenclature) cooperates with a large, predominantly predicted-coiled-coil, protein (Cnn in flies, SPD-5 in worms) to form a scaffold whose assembly is stimulated by the phosphorylation of Cnn/SPD-5 by the mitotic protein kinase Polo/PLK-1. Mitotic centrosome maturation is abolished in the absence of this pathway, and some aspects of Cnn and SPD-5 scaffold assembly have recently been reconstituted in vitro. Vertebrate homologues of Spd-2 (Cep192), Cnn (Cdk5Rap2/Cep215) and Polo (Plk1) also have important roles in mitotic centrosome assembly, indicating that elements of this pathway are likely to be conserved in higher metazoans. In vertebrate cells another centriole and PCM protein, Pericentrin, also has an important role in mitotic centrosome assembly that is dependent upon its phosphorylation by Plk1. Pericentrin can interact with Cep215/Cnn, but in flies the Pericentrin-like-protein (Plp) has a clear, but relatively minor, role in mitotic PCM assembly when compared to Spd-2 and Cnn (Alvarez-Rodrigo, 2019).

Although most of the main players in mitotic centrosome-scaffold assembly appear to have been identified, several fundamental aspects of the assembly process remain mysterious. Cells entering mitosis, for example, contain two mother centrioles that assemble two mitotic centrosomes of equal size. It is unclear how this is achieved, as even a slight difference in the initial size of the two growing centrosomes would be expected to lead to asymmetric centrosome growth-as the larger centrosome would more efficiently compete for scaffolding subunits. The centrioles in fly embryos appear to overcome this problem by constructing the PCM scaffold from the 'inside-out': Spd-2 and Cnn are only incorporated into the scaffold close to the mother centriole, and they then flux outwards to form an expanded scaffold around the mother centriole. In this way, the growing PCM scaffold could ultimately attain a consistent steady-state size-where incorporation around the mother centriole is balanced by loss of the scaffold at the centrosome periphery-irrespective of any initial size difference in the PCM prior to mitosis (Alvarez-Rodrigo, 2019).

A potential problem with this 'inside-out' mode of assembly is that the rate of centrosome growth is limited by the very small size of the centriole. Mathematical modelling indicates that the incorporation of a crucial PCM scaffolding component only around the mother centriole cannot easily account for the high rates of mitotic centrosome growth observed experimentally. To overcome this problem, it has been proposed that centrosome growth is 'autocatalytic', with the centriole initially recruiting a key scaffolding component that can subsequently promote its own recruitment. It has been proposed that Spd-2 and Cnn could form a positive feedback loop that might serve such an autocatalytic function: Spd-2 helps recruit Cnn into the scaffold, and Cnn then helps to maintain Spd-2 within the scaffold, thus allowing higher levels of Spd-2 to accumulate around the mother centriole, which in turn drives higher rates of Cnn incorporation (Alvarez-Rodrigo, 2019).

In worms and vertebrates, SPD-2/Cep192 can help recruit PLK1/Plk1 to centrosomes and Cep192 also activates Plk1 in vertebrates, in part through recruiting and activating Aurora A, another mitotic protein kinase implicated in centrosome maturation. It is suspected, therefore, that in flies Spd-2 might recruit Polo into the centrosome-scaffold to phosphorylate Cnn and so help to generate a positive feedback loop that drives the expansion of the mitotic PCM. In flies, however, no interaction between Polo and Spd-2 has been reported. Indeed, an extensive Y2H screen for interactions between key centriole and centrosome proteins identified interactions between Spd-2 and the mitotic kinases Aurora A and Nek2, and between Polo and the centriole proteins Sas-4, Ana1 and Ana2, but not between Polo and Spd-2. A possible explanation for this result is that Polo/Plk1 is believed to be largely recruited to its many different locations in the cell, including centrosomes, through its Polo-Box-Domain (PBD), which binds to phosphorylated S-S/T(p) motifs. Perhaps any such Polo binding sites in fly Spd-2 were simply not phosphorylated in the Y2H experiments. In support of this possibility, phosphorylated S-S/T(p) motifs in SPD-2/Cep192 have previously been shown to help recruit PLK1/Plk1 to centrosomes in worms, frogs and humans (Alvarez-Rodrigo, 2019).

This study examined the potential role of Spd-2 in recruiting Polo to centrosomes in Drosophila embryos. Like Cnn, Spd-2 is largely unphosphorylated in the cytosol, but is highly phosphorylated at centrosomes, where Spd-2 and Polo extensively co-localise within the pericentriolar scaffold. A Spd-2 fragment containing 19 S-S/T motifs exhibits enhanced binding to the PBD in vitro when it has been phosphorylated by Plk1, but no enhancement is seen if these S-S/T motifs are mutated to T-S/T-a mutation that strongly perturbs PBD binding. This study expressed forms of Spd-2 in vivo in which either all 34 S-S/T motifs, or the 16 most conserved S-S/T motifs, have been mutated to T-S/T to perturb PBD-binding. These mutant Spd-2 proteins are still recruited to mother centrioles, as are Polo and Cnn, and these proteins assemble a PCM scaffold around the mother centriole. Strikingly, however, this PCM scaffold can no longer expand outwards, and centrosome maturation fails. These observations provide strong support for the hypothesis that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that is required to drive the rapid expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).

It was previously proposed that three proteins -- Spd-2, Polo and Cnn -- together form a scaffold that expands around the mother centriole to recruit other PCM components to the mitotic centrosome. The data presented in this study suggests that these three proteins cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic PCM scaffold in fly embryos (Alvarez-Rodrigo, 2019).

The following model is proposed (see Polo and Cnn appear to form a positive feedback loop that drives the expansion of the mitotic PCM scaffold). In interphase cells, Spd-2, Polo and Cnn are recruited around the surface of the mother centriole, but Polo is inactive and Spd-2 and Cnn are not phosphorylated-so no scaffold is assembled. As cells prepare to enter mitosis, centrosomal Spd-2 becomes phosphorylated. In vitro data suggests that Polo is involved in this phosphorylation (via a 'self-priming and binding' mechanism), but other mitotic kinases may also be involved. Phosphorylation allows Spd-2 to form a scaffold that fluxes outwards and that can recruit both Polo (via phosphorylated S-S/T(p) motifs) and Cnn . The active Polo phosphorylates Cnn, allowing it to also form a scaffold. The Spd-2 scaffold is inherently unstable, so it can only accumulate around the mother centriole if it is stabilised by the Cnn scaffold. The Cnn scaffold therefore allows the Spd-2 scaffold to expand outward, increasing Spd-2 levels within the PCM scaffold and allowing Spd-2 to recruit more Cnn and more Polo into the scaffold. This is a classical positive feedback loop in which the Output (the PCM scaffold in toto) directly increases the Input (the Spd-2 scaffold) (Alvarez-Rodrigo, 2019).

If Spd-2 cannot efficiently recruit Polo, as appears to be the case with the Spd-2-ALL and Spd-2-CONS mutants, it can still recruit Cnn, and this is, at least initially, phosphorylated by the pool of Polo that is still present around the mother centriole. The data suggests that this centriolar pool of Polo is not recruited by Spd-2 (at least not via the PBD), and it is suspected that S-S/T(p) motifs in other centriole proteins, such as Sas-4, normally recruit Polo to centrioles. As a result, mutant Spd-2 proteins can still support the assembly of a 'mini-scaffold' around the mother centriole, and this can recruit some PCM and organise some MTs. The mutant Spd-2 scaffold that fluxes outwards from the mother centriole, however, cannot recruit Polo. Therefore the Cnn recruited by the expanding Spd-2 network cannot be phosphorylated, and it cannot form a scaffold to support the expanding Spd-2 network. As a result, the expanding mitotic PCM scaffold rapidly dissipates into the cytosol (Alvarez-Rodrigo, 2019).

Although this mechanism is autocatalytic-as the expanding Spd-2 scaffold allows Polo and Cnn to be recruited into the PCM at an increasing rate-crucially, the mother centriole remains the only source of Spd-2. This potentially explains the conundrum of how mitotic PCM growth is autocatalytic, but at the same time requires the mother centriole. This requirement for centrioles can also potentially explain how two spatially separated centrosomes usually grow their mitotic PCM to the same size, as PCM size may ultimately be determined by how much Spd-2 can be provided by the centrioles, rather than how much PCM was present in the centrosome when maturation was initiated (Alvarez-Rodrigo, 2019).

A key feature of this proposed mechanism is that Cnn cannot recruit itself or Spd-2 or Polo into the scaffold (although it helps to maintain the Spd-2 scaffold recruited by the centriole). If it could do so, mitotic PCM growth would no longer be constrained by the centriole as Cnn could catalyse its own recruitment. Interestingly, although Spd-2 and Cnn are of similar size in flies (1146aa and 1148aa, respectively) Spd-2 has >5X more conserved potential PBD-binding S-S/T motifs than Cnn. Moreover, a similar ratio of conserved sites is found when comparing human Cep192 (1941aa) to human Cep215/Cdk5Rap2 (1893aa), even though the human and fly homologues of both proteins share only limited amino acid identity. Perhaps, these two protein families have evolved to ensure that phosphorylated Spd-2/Cep192 can efficiently recruit Polo/Plk1, whereas phosphorylated Cnn/Cep215 cannot (Alvarez-Rodrigo, 2019).

The data indicates that multiple S-S/T(p) motifs in Spd-2 may be involved in Polo recruitment to the PCM. When only the most conserved motifs are mutated, other motifs in Spd-2 appear to be able to help recruit Polo, as evidenced by the additive effect of the Spd-2-ALL mutant compared to the Spd-2-CONS mutant. This mechanism of multi-site phosphorylation and recruitment could help amplify the maturation process (as the additional Polo recruited would allow Cnn to be phosphorylated at a higher rate) and so contribute to the establishment of the positive feedback loop (Alvarez-Rodrigo, 2019).

Another important feature of this proposed mechanism is that Spd-2 is incorporated into the mitotic PCM at the centriole surface and then fluxes outwards. This Spd-2-flux has so far only been observed in Drosophila embryos and mitotic brain cells. In fly embryos, Cnn also fluxes outwards but, unlike Spd-2, this flux requires MTs and is only observed in embryos. In C. elegans embryos, SPD-5 behaves like Cnn in somatic cells: it does not flux outwards and is incorporated isotropically throughout the volume of the PCM. Moreover, a very recent study found no evidence for an outward centrosomal flux of SPD-2 in worm embryos. Clearly, it will be important to determine whether Spd-2/Cep192 homologues flux outwards in other species and, if so, whether this flux provides the primary mechanism by which the mother centriole influences the growth of the expanding mitotic PCM (Alvarez-Rodrigo, 2019).

In vertebrates, Cep192 serves as a scaffold for Plk1 and also Aurora A-another mitotic protein kinase that plays an important part in centrosome maturation in many species. There appears to be a complex interplay between Cep192, Plk-1 and Aurora A in vertebrates, with Cep192 acting as a scaffold that allows these two important regulators of mitosis to influence each other's activity and centrosomal localisation. Spd-2 clearly plays an important part in recruiting Aurora A to centrosomes in fly cells-although it is unclear if this is direct, as fly and worm Spd-2/SPD-2 both lack the N-terminal region in vertebrate Cep192 that recruits Aurora A. How Aurora A might influence the assembly of the Spd-2, Polo/PLK-1 and Cnn/SPD-5 scaffold remains to be determined, although in worms AIR-1 (the Aurora A homologue) is required to initiate centrosome maturation, but is not required for subsequent PCM growth (Alvarez-Rodrigo, 2019).

Finally, there has been great interest recently in the idea that many non-membrane bound organelles like the centrosome may assemble as 'condensates' formed by liquid-liquid phase separation. In support of this possibility for the centrosome, purified recombinant SPD-5 can assemble into condensates in vitro that have transient liquid-like properties, although they rapidly harden into a more viscous gel- or solid-like phase. Moreover, a mathematical model that describes centrosome maturation in the early worm embryo treats the centrosome as a liquid, and it is from this model that the importance of autocatalysis was first recognised. In vivo, however, the Cnn and SPD-5 scaffolds do not appear to be very liquid-like and fragments of Cnn can assemble into micron-scale assemblies in vitro that are clearly solid- or very viscous-gel-like. The current data suggests that the incorporation of Spd-2 into the PCM only at the surface of the centriole, coupled to an amplifying Spd-2/Polo/Cnn positive feedback loop, could provide an 'autocatalytic' mechanism that functions within the conceptual framework of a non-liquid-like scaffold that emanates from the mother centriole (Alvarez-Rodrigo, 2019).

Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. This study found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. This study has demonstrated that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, this study proposes that two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments (Wang, 2019).

The necessity of sister kinetochores to co-orient toward the same pole for co-segregation at anaphase I differentiates the first meiotic division from the second division. A meiosis-specific mechanism exists that ensures sister chromatid co-segregation by rearranging sister kinetochores, aligning them next to each other and facilitating microtubule attachments to the same pole]. This process is referred to as co-orientation, in contrast to mono-orientation, when homologous kinetochores orient to the same pole. Given the importance of co-orientation in meiosis the mechanism underlying this process is still poorly understood, maybe because many of the essential proteins are not conserved across phyla (Wang, 2019).

Most studies of co-orientation have focused on how fusion of the centromeres and kinetochores is established. In budding yeast, centromere fusion occurs independently of cohesins: Spo13 and the Polo kinase homolog Cdc5 recruit a meiosis-specific protein complex, monopolin (Csm1, Lrs4, Mam1, CK1) to the kinetochore. Lrs4 and Csm1 form a V-shaped structure that interacts with the N-terminal domain of Dsn1 in the Mis12 complex to fuse sister kinetochores. While the monopolin complex is not widely conserved, cohesin-independent mechanisms may exist in other organisms. A bridge between the kinetochore proteins MIS12 and NDC80 is required for co-orientation in maize. In contrast, cohesins are required for co-orientation in several organisms. The meiosis-specific cohesin Rec8 is indispensable for sister centromere fusion in fission yeast and Arabidopsis. Cohesin is localized to the core-centromere in fission yeast and mice. In Drosophila melanogaster oocytes, cohesins (SMC1/SMC3/SOLO/SUNN) establish cohesion in meiotic S-phase and show an enrichment that colocalizes with centromere protein CID/CENP-A. Like fission yeast and mouse, Drosophila may require high concentrations of cohesins to fuse sister centromeres together for co-orientation during meiosis (Wang, 2019).

In mice, a novel kinetochore protein, Meikin, recruits Plk1 to protect Rec8 at centromeres. Although poorly conserved, Meikin is proposed to be a functional homolog of Spo13 in budding yeast and Moa1 in fission yeast. They all contain Polo-box domains that recruit Polo kinase to centromeres. Loss of Polo in both fission yeast (Plo1) and mice results in kinetochore separation, suggesting a conserved role for Polo in co-orientation. In fission yeast, Moa1-Plo1 phosphorylates Spc7 (KNL1) to recruit Bub1 and Sgo1 for the protection of centromere cohesion in meiosis I. These results suggest the mechanism for maintaining sister centromere fusion involves kinetochore proteins recruiting proteins that protect cohesion. However, how centromere cohesion is established prior to metaphase I, and how sister centromere fusion is released during meiosis II, still needs to be investigated (Wang, 2019).

Previous work has found that depletion of the kinetochore protein SPC105R (KNL1) in Drosophila oocytes results in separated centromeres at metaphase I, suggesting a defect in sister centromere fusion. Thus, Drosophila SPC105R and fission yeast Spc7 may have conserved functions in co-orientation (Radford, 2015). This study has identified a second Drosophila protein required for sister-centromere fusion, Protein Phosphatase 1 isoform 87B (PP1-87B). However, sister centromere separation in SPC105R and PP1-87B depleted Drosophila oocytes occurs by different mechanisms, the former is Separase dependent and the latter is Separase independent. Based on these results, a model is proposed for the establishment, protection and release of co-orientation. Sister centromere fusion necessary for co-orientation is established through cohesins that are protected by SPC105R. Subsequently, PP1-87B maintains co-orientation in a cohesin-independent manner by antagonizing stable kinetochore-microtubule (KT-MT) interactions. The implication is that the release of co-orientation during meiosis II is cohesin-independent and MT dependent. A surprising interaction was found between PP1-87B and C(3)G, the transverse element of the synaptonemal complex (SC), in regulating sister centromere separation. Overall, these results suggest a new mechanism where KT-MT interactions and centromeric SC regulate sister kinetochore co-orientation during female meiosis (Wang, 2019).

Polo regulates Spindly to prevent premature stabilization of kinetochore-microtubule attachments

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, this study has identified a strong genetic interaction between Polo and the Rod-<ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. It is proposed that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. These findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation (Barbosa, 2020).

To ensure the fidelity of chromosome segregation, sister kinetochores (KTs) mediate the attachment of chromosomes to microtubules (MTs) of opposite spindle poles (amphitelic attachments). However, the initial contact of KTs with MTs is stochastic and consequently erroneous attachments-syntelic (chromosome bound to MTs from the same spindle pole) or merotelic (same KT bound to MTs from opposite poles)-can be formed during early mitosis. Thus, accurate mitosis requires a tight regulation of KT-MT turnover so mistakes are prevented or corrected and amphitelic end-on interactions are stabilized. This relies heavily on the activity of two conserved mitotic kinases, Aurora B and Polo/Plk1. Aurora B promotes the destabilization of KT-MT interactions mainly through phosphorylation of proteins of the KMN network (KNL1/Spc105, Mis12 and Ndc80), which decreases their affinity for MTs. Interestingly, it has been shown that the RZZ complex (Rod, ZW10 and Zwilch) is able to interact with Ndc80 N-terminal tail and prevent the adjacent calponin homology (CH) domain from binding to tubulin (Cheerambathur, 2013). This Aurora B-independent destabilizing mechanism is proposed to prevent Ndc80-mediated binding when KTs are laterally attached, hence reducing the potential for merotely during early mitosis. The RZZ additionally recruits Spindly and the minus end-directed motor dynein to KTs, thus providing the means to relieve its inhibitory effect over KT-MT attachments, as well as to ensure the timely removal of spindle assembly checkpoint (SAC) proteins from KTs. However, it remains unclear how RZZ removal by Spindly-dynein is coordinated with end-on attachment formation (Barbosa, 2020).

Polo/Plk1 activity is implicated in both stabilization and destabilization of KT-MT attachments. While the contribution to the former function has been attributed to PP2A-B56 phosphatase recruitment through Plk1-dependent BubR1 phosphorylation, the mechanism underlying Polo/Plk1 destabilizing activity remains unclear. Interestingly, Polo/Plk1 KT localization and activity decrease from early mitosis to metaphase, concurrent with an increase in KT-MT stability. Moreover, high Plk1 activity at KTs was shown to correlate with decreased stability of KT-MT attachments during prometaphase, but the underlying molecular mechanisms have only been marginally addressed (Barbosa, 2020).

This study describes the mitotic effect of expressing a constitutively active Polo-kinase mutant (PoloT182D) in Drosophila neuroblasts and cultured S2 cells. The expression of PoloT182D causes persistent KT-MT instability and congression defects, extends mitotic timing associated with SAC activation and increases chromosome mis-segregation. A small-scale candidate-based RNAi screen was designed to identify partners/pathways that are affected by constitutive Polo activity in the Drosophila eye epithelium. The screen revealed that downregulation of the RZZ subunit Rod rescues the defects resulting from PoloT182D expression. PoloT182D causes permanent accumulation of the RZZ complex at KTs, which is associated with a delay in achieving stable biorientation. Accordingly, Rod depletion rescues the time required for establishing end-on KT-MT attachments and for chromosome congression. This study further demonstrates that Polo phosphorylates the dynein-adaptor Spindly to decrease its affinity for the RZZ. This in turn prevents dynein-dependent stripping of RZZ from KTs, hence causing a delay in the formation of stable end-on attachments. The findings provide a mechanism for the destabilizing action of Polo/Plk1 over KT-MT attachments. A model is proposed in which Polo/Plk1 activity fine-tunes the RZZ-Spindly-dynein module throughout mitosis to ensure the fidelity of KT-MT attachments and chromosome segregation (Barbosa, 2020).

KT-MT attachments at metaphase must be sufficiently stable to satisfy the spindle assembly checkpoint and sustain chromatid segregation during anaphase. On the other hand, during prometaphase, MTs must be able to rapidly detach from KTs to allow efficient correction of erroneous attachments. How KTs regulate the balance of MTs stabilizing and destabilizing forces during successive mitotic stages has remained unclear. This study shows that Polo kinase plays a critical role in this process through control of the RZZ-Spindly-dynein module at KTs. Polo-mediated phosphorylation of Spindly on Ser499 results in a transient increase in RZZ accumulation at KTs, which inhibits stable end-on attachments and likely minimizes merotely in early mitosis. However, permanent Spindly Ser499 phosphorylation is deleterious for mitotic fidelity since it prevents stable KT biorientation and timely chromosome congression (Barbosa, 2020).

Polo/Plk1 has been implicated in the stabilization of KT-MT attachments. Intriguingly, however, attachments are most stable during metaphase, when Polo/Plk1 activity is reduced. Maintaining Polo active in Drosophila larval neuroblasts markedly decreases the stability of KT-MT interactions, which is in line with previous observations in RPE-cultured cells. It is important to mention that insc-GaL4- driven expression of PoloWT and PoloT182D consistently yielded higher levels of the latter protein. This, however, unlikely explains the different phenotypic consequences observed in these neuroblasts, as analogous experiments with S2 cells expressing equivalent levels of PoloWT and PoloT182D mimic the decrease in the efficiency of chromosome congression and KT-MT stability when constitutively active Polo is expressed. Moreover, previous work has shown that Drosophila S2 cells depleted of Polo accumulated hyperstable attachments and that this phenotype was not exclusively attributed to reduced Aurora B activity. A requirement for Polo in fine-tuning the RZZ-Spindly-dynein axis offers a mechanistic explanation for these observations. During early mitosis, high levels of active Polo at KTs ensure that as soon as Spindly is recruited to RZZ, it is efficiently phosphorylated on Ser499. This promptly reduces Spindly affinity towards Zwilch, sensitizing RZZ-uncoupled Spindly for dynein-mediated transport away from KTs. As a result, the RZZ complex is retained at KTs to levels that normally inhibit the formation of stable end-on attachments by the Ndc80 complex (Cheerambathur, 2013) and maintain the SAC signalling active . Rod interacts with the basic tail of Ndc80 and, in this way, precludes binding of MTs to the calponin homology domain of Ndc80 (Cheerambathur, 2013). Thus, the conversion of lateral attachments preferentially formed at early stages of mitosis into stable amphitelic interactions that are essential for faithful chromosome segregation requires the relief of this Rod-mediated inhibitory mechanism. Evidence is provided that a decrease in Polo activity and, consequently, in Spindly phosphorylation, is critical for this transition by allowing the RZZ to fully engage with the Spindly-dynein complex and to be stripped from KTs. This raises the question of how and when Polo activity and Ser499 phosphorylation are antagonized to allow timely formation of stable end-on attachments. PP2A-B56 phosphatase may have a role in this process, since impairing its association with BubR1 was recently shown to dramatically increase the frequency of laterally attached KTs in human cells. However, because BubR1-PP2A-B56 is already present at high levels on early mitotic KTs, it was reason that additional mechanisms must operate to prevent premature end-on conversion. It is plausible that the switch is determined by the levels of cyclin A, which have been shown to function as a timer in prometaphase to destabilize attachments and facilitate error correction. Since Cdk1/CycA is able to phosphorylate human Spindly in vitro, it is hypothesized that this phosphorylation primes Spindly for Polo binding and increases Ser499 phosphorylation to levels that surpass the opposing phosphatase activity. As mitosis progresses, degradation of Cyclin A tips the balance towards Ser499 dephosphorylation, hence favouring stabilization of end-on attachments. This concurs with an increase in tension across KTs that allows the recruitment of PP1, whose role in Polo T-loop dephosphorylation has been described (Barbosa, 2020).

Although the Polo-phosphorylation site in Drosophila Spindly is not conserved in vertebrates, additional residues conforming to Polo/Plk1 consensus signature are present within the same domain, hinting that an analogous regulatory mechanism may take place in these organisms. Interestingly, Ser499 lies within motif that is conserved among different dynein-adaptors. Two other conserved domains have also been recently described for a number of adaptors and shown to act as regulatory modules involved in the interaction with dynein. Thus, it is envision that the motif identified in this study might provide an additional level of regulation in controlling dynein-adaptor complex formation (Barbosa, 2020).

The results suggest that Polo-mediated phosphorylation of Spindly on Ser499 uncouples dynein-mediated transport of the RZZ complex from Spindly. Moreover, it is proposed that phosphorylation of Ser499 causes Spindly C-terminal domain to elicit a negative regulatory action over the N-terminus Zwilch binding domain. In line with these results, it has been recently shown that intramolecular interactions occur within Spindly, causing it to fold on itself at different regions (Sacristan, 2018). Spindly C-terminal region could be involved in facilitating these interactions since it is thought to be of disordered nature (Sacristan, 2018). This structural organization resembles that of BicD/BicD2, a dynein-adaptor which is predicted to share with Spindly a similar mechanism of interaction with dynein. It is therefore noteworthy that Polo has been shown to activate BicD-dynein transport during oogenesis. Furthermore, several point mutations in BicD/BicD2 were shown to hyperactivate dynein for cargo transport. It will be interesting to establish whether Spindly Ser499 phosphorylation could also impact on dynein complex motility/processivity (Barbosa, 2020).

Long-lasting Polo activation or permanent Spindly Ser499 phosphorylation stalls KTs in labile interactions with MTs. The data confirm a destabilizing role for Polo in KT-MT attachments which has also been shown to operate through the control the kinase exerts over the recruitment and activation of Aurora B and the MT depolymerizing motor Kif2b . Hence, high levels of active Polo in early mitosis ensure efficient correction of merotelic and syntelic attachments, errors that typically occur upon nuclear envelope breakdown as a result of stochastic interactions between KTs and spindle MTs. Paradoxically, Plk1 activity has also been implicated in stabilization of KT-MT attachments through phosphorylation of BubR1. A model is envisioned where these apparently antagonistic Polo-directed inputs are not mutually exclusive but rather cooperate to establish proper attachments. Phosphorylation of BubR1 by Polo/Plk1 in prometaphase promotes the accumulation of PP2A-B56, which opposes Aurora B destabilizing phosphorylations on Ndc80. This is important to allow binding of MTs to the Ndc80 complex during the end-on conversion process, tipping the balance against the KT-MT destabilizing environment, particularly when Cyclin A levels drop. The observation that disrupting Plk1 activity rescues the attachment defects otherwise generated by depletion of PP2A-B56 strongly argues in favour of this integrated model for Polo-regulated stabilizing and destabilizing forces (Barbosa, 2020).

In summary, these findings demonstrate that the RZZ-Spindly-dynein module is tightly regulated by Polo kinase to ensure accurate chromosome segregation. Spindly phosphorylation by Polo on early mitotic KTs ensures RZZ-mediated inhibition of end-on interactions, hence preventing premature stabilization of erroneous attachments. As mitosis progresses, decreased Polo-kinase activity and concurrent Spindly dephosphorylation render the RZZ prone for removal from KTs by Spindly-dynein. This alleviates RZZ antagonism of MT binding by the Ndc80 complex, thus allowing timely conversion of labile lateral interactions into stable amphitelic attachments ensuring proper sister chromatid segregation (Barbosa, 2020).

Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, this study shows in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, it is proposed that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation (Gallaud, 2020).

Centrosome asymmetry has previously been described to occur in asymmetrically dividing Drosophila neural stem cells (neuroblasts), manifested in biased interphase MTOC activity and asymmetric localization of the centrosomal proteins Cnb, Plp, and Polo and PCM proteins like Centrosomin. This study has shown that neuroblast centrosomes become intrinsically asymmetric by dynamically enriching centriolar proteins such as Cnb and Polo on the young daughter centriole during mitosis. This establishment of centriolar asymmetry is tightly linked to centriole-to-centrosome also called mitotic centriole conversion. In early prophase, Cnb and Polo colocalize on the existing mother centriole of the apical centrosome but from late prometaphase onward, Cnb is exclusively and Polo predominantly localized on the daughter centriole. Mechanistically, these dynamic localization changes could entail a direct or indirect translocation of Cnb and Polo from the mother to the daughter centriole. This model is partially supported for Cnb with FRAP data. Interestingly, Cnb behaves differently on the basal centrosome: the existing mother centriole does not contain any Cnb, appearing only on the forming daughter centriole in late prophase. This suggests a direct recruitment mechanism, which could also apply to the apical centrosome from anaphase onward. 3D-SIM, FRAP, and live-cell imaging data combined are most consistent with a model proposing that on the apical centrosome, a small pool of Cnb transfers from the mother to the daughter centriole during early mitosis. From anaphase onward, and from late prophase onward on the basal daughter centriole, Cnb levels increase through the recruitment of Cnb that was not previously associated with the mother centriole (Gallaud, 2020).

Cnb is phosphorylated by the mitotic kinase Polo and Polo-dependent phosphorylation of Cnb is necessary for its timely localization during mitosis. Interestingly, the data further suggest that Polo, which also becomes enriched on the daughter centriole during mitosis, is co-dependent with Cnb, while also requiring Wdr62. Polo's involvement in mitotic centriole conversion further suggests that the same molecular machinery cooperatively converts a maturing centriole into a centrosome for the next cell cycle while simultaneously providing it with its unique molecular identity (Gallaud, 2020).

The mechanisms generating 2 molecularly distinct centrioles during mitosis seem to directly influence the centrosome's MTOC activity in interphase; the 'Cnb+, high Polo' daughter centriole will retain MTOC activity during interphase whereas the 'Cnb-, low Polo' mother centriole, separates from its daughter in early interphase and becomes inactive. This model is in agreement with bld10 (Cep135) or plp mutants, which fail to down-regulate Polo from the mother centriole, resulting in the formation of 2 active interphase MTOCs. This is further supported by mislocalization data, showing that optogenetic manipulation of Polo and Cnb asymmetry specifically during mitosis impacts MTOC activity in the subsequent interphase. However, it cannot be excluded that MTOC asymmetry is also controlled independently of mitotic centrosome asymmetry establishment because optogenetic interphase manipulations of Polo and Cnb alone can also perturb biased MTOC activity (Gallaud, 2020).

Loss of Wdr62 or Cnb also affects asymmetric centriolar Polo localization. Yet, interphase centrosomes lose their activity in these mutants. wdr62 mutants and cnb RNAi neuroblasts both show low Polo levels in interphase. It is thus hypothesized that in addition to an asymmetric distribution, Polo levels must remain at a certain level to maintain interphase MTOC activity; high symmetric Polo results in 2 active interphase MTOCs, whereas low symmetric Polo results in the formation of 2 inactive centrosomes. Indeed, the optogenetic experiment triggered an increase in centriolar Polo levels upon blue-light induction, suggesting that both Polo levels and distribution influence MTOC activity. This hypothesis is strengthened by Cnn's capacity to oligomerize and form a scaffold, supporting PCM assembly upon phosphorylation by Polo. The more Polo is recruited, the more stable is the Cnn scaffold, supporting MTOC activity (Gallaud, 2020).

Centrobin is also enriched on the daughter centriole in mammals and preferentially becomes incorporated into the newly assembled daughter centriole in late G1 or early S phase. Centrobin remains localized at the daughter centrioles throughout the cell cycle. It is of interest to note that in mammalian cells, Centrobin becomes enriched on daughter centrioles during the G1-S transition and not during centriole-to-centrosome conversion. It is tempting to speculate that other kinases and mechanism regulate this translocation (Gallaud, 2020).

It was also observed that Cnb and Plp localize in a mutually exclusive manner, with Cnb localizing to the daughter centriole and Plp remaining on the mother centriole. In mammalian cells, similar mutual exclusion between the centriolar proteins Cep120 or Neurl4 and PCM proteins has been observed (Gallaud, 2020).

Taken together, the results reported in this study are consistent with a model, proposing that the establishment of 2 molecularly distinct centrioles is primed during mitosis and contributes to biased MTOC activity in the subsequent interphase. Wild-type neuroblasts unequally distribute a given pool of Cnb and Polo protein between the 2 centrioles so that the centriole inheriting high amounts of Cnb and Polo will retain MTOC activity. Furthermore, the dynamic localization of Polo and Cnb provides a molecular explanation for why the daughter-centriole-containing centrosome remains tethered to the apical neuroblast cortex and is being inherited by the self-renewed neuroblast. It remains to be tested why neuroblasts implemented such a robust machinery to asymmetrically segregate the daughter-containing centriole to the self-renewed neuroblast. More refined molecular and behavioral assays will be necessary to elucidate the developmental and postdevelopmental consequences of biased centrosome segregation. The tools and findings reported in this study will be instrumental in targeted perturbations of intrinsic centrosome asymmetry with spatiotemporal precision in defined neuroblast lineages (Gallaud, 2020).

Finally, the observations reported in this study further raise the tantalizing possibility that centriolar proteins also dynamically localize in other stem cells, potentially providing a mechanistic explanation for the differences in centriole inheritance across different stem cell systems (Gallaud, 2020).

Ana1 helps recruit Polo to centrioles to promote mitotic PCM assembly and centriole elongation

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and it has been shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. This study shows that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. It is concluded that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly (Alvarez-Rodrigo, 2021).

Polo kinase (PLK1 in mammals) is an important cell cycle regulator. During mitosis, it is recruited to several locations in the cell -- such as centrosomes, kinetochores and the cytokinesis apparatus -- where it performs multiple functions. PLK1 is usually recruited to these locations by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs in target proteins. Mutating the first serine in the PBD-binding motif to threonine strongly reduces PBD binding in vitro and in vivo (Alvarez-Rodrigo, 2021).

PLK1 has several key functions at centrosomes. These organelles are important microtubule (MT) organising centres that form around a pair of centrioles (comprising a mother and daughter centriole) when the mother recruits a matrix of pericentriolar material (PCM) around itself. During interphase, centrosomes organise relatively little PCM, but as cells prepare to enter mitosis the PCM expands dramatically in a process termed centrosome maturation. PLK1 is an essential driver of this process, and several PCM proteins have been identified as PLK1 targets. In vertebrate cells, PLK1 phosphorylates pericentrin, which cooperates with CDK5RAP2 (also known as Cep215) to promote mitotic PCM assembly, whereas in flies and worms Polo/PLK1 kinases phosphorylate Cnn and SPD-5 (functional homologues of CDK5RAP2), respectively, which allows these proteins to assemble into a PCM scaffold around the mother centriole that recruits other PCM proteins (Alvarez-Rodrigo, 2021).

Towards the end of mitosis, the mother and daughter centrioles disengage from each other. PLK1 is essential for disengagement and also for the subsequent maturation of the daughter centriole into a new mother centriole that is itself capable of duplicating and organising PCM. The old mother (OM) and new mother (NM) centrioles then both duplicate during S phase by nucleating the assembly of a daughter centriole on their side. PLK1 is not essential for centriole duplication per se, but it is required for the growth of the centriole MTs that occurs during G2, at least in human cells, and for the subsequent maturation of the daughter centriole into a new mother centriole. After duplication in S phase, the two centrosomes (each now comprising a duplicated centriole pair) are held together by a linker, and PLK1 also helps disassemble this linker to promote centrosome separation as cells prepare to enter mitosis (Alvarez-Rodrigo, 2021).

How PLK1 is recruited to centrosomes to execute its multiple functions is largely unclear, although this recruitment appears to be dependent on the PBD. In vertebrate systems, Cep192 is required for centrosome maturation and it is phosphorylated by Aurora A (also known as AURKA) to create PBD-binding sites that recruit PLK1; this promotes the activation of both kinases at the centrosome. The fly and worm homologues of Cep192, Spd-2 and SPD-2, respectively, are concentrated at centrioles and centrosomes, and their phosphorylation also helps recruit Polo/PLK1 kinases to the mitotic PCM to phosphorylate Cnn in flies and SPD-5 in worms. In fly embryos, Spd-2, Polo and Cnn have been proposed to form a positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. In this scenario, Spd-2 starts to be phosphorylated at centrioles as cells prepare to enter mitosis, and this allows Spd-2 to form a scaffold that can recruit other PCM proteins and that fluxes outwards from the mother centriole. The Spd-2 scaffold itself is weak, but it can recruit Polo and Cnn; the recruited Polo phosphorylates Cnn, which then forms a Cnn scaffold that recruits other PCM components and strengthens the Spd-2 scaffold. This allows more Spd-2 to accumulate around the centriole, which in turn drives the recruitment of more Polo and Cnn - so forming a positive feedback loop. In this way, Spd-2 recruits Polo and Cnn to the PCM to help drive centrosome maturation in flies (Alvarez-Rodrigo, 2021).

If Drosophila Spd-2 cannot efficiently recruit Polo (because all its S-S/T motifs have been mutated to T-S/T) Polo recruitment to the PCM is dramatically reduced, but Polo is still strongly recruited to the mother centriole, indicating that other proteins must help recruit Polo to centrioles. The centriole protein Sas-4 is phosphorylated by Cdk1 during mitosis on threonine 200 (T200), creating a PBD-binding site that recruits Polo to newly formed daughter centrioles. This allows the daughter to recruit Asl (Cep152 in humans), which allows the daughter to mature into a new mother that can duplicate and organise PCM - since Asl is required for both of these processes. Although the single PBD-binding site in Sas-4 recruits Polo to mother centrioles, it is suspected that other proteins must also be required. This study attempted to identify such proteins by mutating all the S-S/T motifs to T-S/T in several candidates. The centriole protein Ana1 (Cep295 in humans) was found to normally help recruit Polo to mother centrioles. Ana1 and Cep295 are required for centriole maturation, and in flies Ana1 helps recruit and/or maintain Asl at new mother centrioles. Thus, flies lacking Ana1 lack centrioles, centrosomes and cilia, presumably because the centrioles cannot duplicate without Ana1 as they cannot recruit Asl. This study shows that centrioles that do not efficiently recruit Polo via Ana1 can still recruit Sas-4, Cep135 and Asl, and can still duplicate, disengage and organise cilia, but they cannot efficiently recruit mitotic PCM or elongate during G2. It is proposed that Ana1 recruits Polo to centrioles specifically to promote centriole elongation in G2 and mitotic PCM assembly (Alvarez-Rodrigo, 2021).

Polo has many important functions at centrioles and centrosomes, and previous work has shown that it is initially recruited to newborn centrioles in flies when Cdk1 phosphorylates the Sas-4 T200 S-T motif during mitosis. This initial recruitment of Polo is important to allow the newborn centrioles to subsequently mature into mothers that can recruit Asl and so duplicate and recruit mitotic PCM. This study showed that the centriole protein Ana1 also plays an important part in recruiting Polo to mother centrioles. The data suggests that Ana1 can recruit Polo directly and that Polo itself can phosphorylate Ana1 at several S-S/T motifs to 'self-prime' its own recruitment. It cannot be excluded that other protein kinases may prime these S-S/T motifs, or that Ana1 could recruit Polo to centrioles indirectly in ways that are disrupted when the S-S/T motifs are mutated to T-S/T. Regardless of mechanism, the Ana1-dependent centriolar pool of Polo appears to be required to drive efficient mitotic PCM expansion and centriole elongation in G2 (Alvarez-Rodrigo, 2021).

Although Ana1 helps recruit and/or maintain Asl at centrioles, and therefore is essential for both mitotic PCM recruitment and centriole duplication, this function of Ana1 does not appear to require the ability to recruit Polo. Thus, Ana1-S34T centrioles recruit and maintain normal levels of Asl (and of Cep135, as well as slightly increased levels of Sas-4) and can duplicate normally. This is in contrast to the situation with Sas-4, where T200 phosphorylation is required for proper Asl recruitment and so for both centriole duplication and mitotic PCM assembly. Presumably, the Polo recruited by Sas-4 is either sufficient for Asl recruitment, or it phosphorylates centriole substrates other than Ana1 to promote Asl recruitment. Interestingly, PLK1 is also essential for efficient centriole disengagement, but neither the Ana1-S34T nor Sas-4-T200 mutations appear to perturb this process, indicating that a separate pathway must recruit Polo to centrioles to drive centriole disengagement. Centrosome separation in G2 is also normally dependent on PLK1, and centrosomes/duplicated centriole pairs were often observed that failed to separate properly in embryos expressing Ana1-S34T. As these centriole pairs almost always organised very little PCM, however, it is suspected that this defect may be an indirect consequence of the failure to properly recruit PCM, rather than a direct consequence of the inability of Ana1 to recruit Polo (Alvarez-Rodrigo, 2021).

These new findings further support the hypothesis that centrioles activate a Spd-2-Polo-Cnn positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. A key feature of this model is that Spd-2 can only be phosphorylated to initiate scaffold assembly at the surface of the mother centriole, and the phosphorylated Spd-2 then fluxes outwards away from the centriole: the Spd-2-Polo-Cnn scaffold itself cannot phosphorylate and/or recruit new Spd-2 into the scaffold. This is important, as it can explain why the mother centriole is required to drive efficient mitotic PCM assembly, why the size of the centriole influences the size of the mitotic PCM and why centrioles are constantly required to drive the growth of the mitotic PCM. All of these findings can be explained if the mother centriole is the only source of the phosphorylated Spd-2 scaffold. The observation that the pool of Polo recruited by Ana1, which unlike Spd-2, is not a PCM component and is restricted to the centriole - is required for the efficient expansion of the PCM demonstrates that the PCM-associated pool of Polo (recruited by Spd-2) is not sufficient to drive efficient PCM expansion on its own. It is important to stress, however, that so far an outward flux of Spd-2 from the centriole has only been observed in fly embryos and cells and has not been detected for SPD-2 in C. elegans embryos\. Clearly it will be important to establish whether such a Spd-2 or Cep192 flux exists in other species (Alvarez-Rodrigo, 2021).

The ability of Ana1 to recruit Polo also appears to be required for centriole elongation during G2. In human cells, PLK1 is required for this process, although a previous study did not report any change in centriole length after long-term Polo-inhibition in fly spermatocytes. Clearly more work is required to establish whether Polo recruitment by Ana1 has a role in G2 centriole elongation in flies, as the current work suggests, and, if so, what Polo's relevant substrates are at the centriole distal end (Alvarez-Rodrigo, 2021).

Finally, it is noted that both the Ana1/Cep295 and Spd-2/Cep192 protein families have a relatively high density of potential PBD-binding sites (S-S/T motifs) when compared to several other centriole and centrosome proteins. This suggests that these proteins might have evolved to function as scaffolds that amplify Polo levels at specific locations within the cell during mitosis. It will be interesting to examine whether other proteins with a high density of potential PBD-binding domains serve a similar function at other locations within the mitotic cell. The strategy of mutating all S-S/T motifs to T-S/T in candidate proteins may be a good way of testing this possibility as, for both Ana1 and Spd-2 at least, the S-to-T substitutions seem to specifically impair Polo-recruitment without more generally perturbing the function of the proteins or centriole/centrosome structure (Alvarez-Rodrigo, 2021).


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: 5 December 2023 

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