Plk4 triggers autonomous de novo centriole biogenesis and maturation

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.     

Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome

Both gain and loss of function studies have identified the Polo-like kinase Plk4/Sak as a crucial regulator of centriole biogenesis, but the mechanisms governing centrosome duplication are incompletely understood. In this study, we show that the pericentriolar material protein, Cep152, interacts with the distinctive cryptic Polo-box of Plk4 via its N-terminal domain and is required for Plk4-induced centriole overduplication. Reduction of endogenous Cep152 levels results in a failure in centriole duplication, loss of centrioles, and formation of monopolar mitotic spindles. Interfering with Cep152 function prevents recruitment of Plk4 to the centrosome and promotes loss of CPAP, a protein required for the control of centriole length in Plk4-regulated centriole biogenesis. Our results suggest that Cep152 recruits Plk4 and CPAP to the centrosome to ensure a faithful centrosome duplication process.     

Centriole duplication

In interphase and mitosis, centrosomes play a major role in the spatial organization of the microtubule network. Alterations in centrosome number and structure are associated with genomic instability and occur in many cancers. Centrosome duplication is controlled by centriole replication. In most dividing animal cells, centrioles duplicate only once per cell cycle at a site adjacent to existing centrioles. The conserved protein kinase Polo-like kinase 4 (Plk4) has a key role in controlling centriole biogenesis. Overexpression of Plk4 drives centrosome amplification, leading to genomic instability and the formation of tumors in flies. By contrast, haploinsufficiency of Plk4 causes cytokinesis failure leading to an increased incidence of tumors in mice. Recent studies have shown that Plk4 is a low abundance protein whose stability is linked to the activity of the enzyme. We discuss how this autoregulatory feedback loop acts to limit the damaging effects caused by too much or too little Plk4.     

Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability

Accurate control of the number of centrosomes, the major microtubule-organizing centers of animal cells, is critical for the maintenance of genome integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation, and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material, and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication, and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Using chemical genetics, we show that kinase-active Plk4 is inherently unstable and targeted for degradation. Plk4 is shown to multiply self-phosphorylate within a 24–amino acid phosphodegron. Phosphorylation of multiple sites is required for Plk4 instability, indicating a requirement for a threshold level of Plk4 kinase activity to promote its own destruction. We propose that kinase-mediated, autoregulated instability of Plk4 self-limits Plk4 activity so as to prevent centrosome amplification.     

Plk2 regulated centriole duplication is dependent on its localization to the centrosome and a functional polo-box domain

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell-cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G1/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G1 phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated down-regulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.     

Polo-like kinase-2 is required for centriole duplication in mammalian cells

Centriole duplication initiates at the G1-to-S transition in mammalian cells and is completed during the S and G2 phases. The localization of a number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling the centriole duplication cycle. Here we show that the human Polo-like kinase 2 (Plk2) is activated near the G1-to-S transition of the cell cycle. Endogenous and overexpressed HA-Plk2 localize with centrosomes, and this interaction is independent of Plk2 kinase activity. In contrast, the kinase activity of Plk2 is required for centriole duplication. Overexpression of a kinase-deficient mutant under S-phase arrest blocks centriole duplication. Downregulation of endogenous Plk2 with small hairpin RNAs interferes with the ability to reduplicate centrioles. Furthermore, centrioles failed to duplicate during the cell cycle of human fibroblasts and U2OS cells after overexpression of a Plk2 dominant-negative mutant. These results show that Plk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1-to-S phase transition.     

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2A(Twins)) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.     

Centrosome duplication: of rules and licenses

Most microtubule arrays in animal cells, including the bipolar spindle required for cell division, are organized by centrosomes. Thus, strict control of centrosome numbers is crucial for accurate chromosome segregation. Each centrosome comprises two centrioles, which need to be duplicated exactly once in every cell cycle. Recent work has begun to illuminate the mechanisms that regulate centriole duplication. First, genetic and structural studies concur to delineate a centriole assembly pathway in Caenorhabditis elegans. Second, the protease Separase, previously known to trigger sister chromatid separation, has been implicated in a licensing mechanism that restricts centrosome duplication to a single occurrence per cell cycle. Finally, Plk4 (also called Sak), a member of the Polo kinase family, has been identified as a novel positive regulator of centriole formation.     

Cep152 interacts with Plk4 and is required for centriole duplication

Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1-217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation.     

DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement

Centrioles form cilia and centrosomes, organelles whose dysfunction is increasingly linked to human disease. Centriole duplication relies on a few conserved proteins (ZYG-1/Sak/Plk4, SAS-6, SAS-5/Ana2, and SAS-4), and is often initiated by the formation of an inner "cartwheel" structure. Here, we show that overexpressed Drosophila Sas-6 and Ana2 coassemble into extended tubules (SAStubules) that bear a striking structural resemblance to the inner cartwheel of the centriole. SAStubules specifically interact with centriole proximal ends, but extra DSas-6/Ana2 is only recruited onto centrioles when Sak/Plk4 kinase is also overexpressed. This extra centriolar DSas-6/Ana2 induces centriole overduplication and, surprisingly, increased centriole cohesion. Intriguingly, we observe tubules that are structurally similar to SAStubules linking the engaged centrioles in normal wild-type cells. We conclude that DSas-6 and Ana2 normally cooperate to drive the formation of the centriole inner cartwheel and that they promote both centriole duplication and centriole cohesion in a Sak/Plk4-dependent manner.     

Plk4-induced centriole biogenesis in human cells

We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.     

Binding of STIL to Plk4 activates kinase activity to promote centriole assembly

Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.     

SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis

Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.     

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.     

Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway

The presence of supernumerary centrosomes in cells infected with Chlamydia trachomatis may provide a mechanism to explain the association of C. trachomatis genital infection with cervical cancer. We show that the amplified centrosomal foci induced during a chlamydial infection contain both centriolar and pericentriolar matrix markers, demonstrating that they are bona fide centrosomes. As there were multiple immature centrioles but approximately one mature centriole per cell, aborted cytokinesis alone cannot account for centrosome amplification during a chlamydial infection. Production of supernumerary centrosomes required the kinase activities of Cdk2 and Plk4, which are known regulators of centrosome duplication, and progression through S-phase, which is the stage in the cell cycle when duplication of the centrosome occurs. These requirements indicate that centrosome amplification during a chlamydial infection depends on the host centrosome duplication pathway, which normally produces a single procentriole from each template centriole. However, C. trachomatis induces a loss of numerical control so that multiple procentrioles are formed per template.     

Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations

Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP–Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.     

Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.     

Reproductive maternal centrosomes are cast off into polar bodies during maturation division in starfish oocytes

Animal egg inherits a maternal centrosome from the meiosis-II spindle and sperm can introduce another centrosome at fertilization. It has been believed that in most animals only the sperm centrosome provides the division poles for mitosis in zygotes. This uniparental (paternal) inheritance of the centrosome must depend on the loss of the maternal centrosome. In starfish, suppression of polar body (PB) extrusion is a prerequisite for induction of parthenogenesis (Washitani-Nemoto et al. (1994) Dev. Biol. 163, 293-301), suggesting that the centrosomes cast off into PBs have reproducing capacity. Due to the absence of centriole duplication in meiosis II of starfish oocytes, each centrosome of a meiosis-II spindle has only one single centriole, whereas in meiosis I each has a pair of centrioles (Sluder et al. (1989) Dev. Biol. 131, 567-579; Kato et al. (1990) Dev. Growth Differ. 32, 41-49). Hence, the first PB (PB1) has two centrioles, whereas the second PB (PB2) and the mature egg have only one centriole, respectively. The present study examined the reproductive capacity of PB centrosomes by transplanting them into artificially activated eggs, and then the recipient egg nucleus with the surrounding cytoplasm was removed. A transplanted PB2 centrosome with a single centriole formed a monopolar spindle at the first mitosis, followed by formation of a bipolar spindle in the next mitosis, leading to actual cleavage and subsequent development. This proves the reproducing capacity of the single centriole in the PB2 centrosome. The behavior of the transplanted PB1 centrosome was exactly the same as in the PB2 centrosome, in spite of the difference in the number of centrioles. These results clearly show that four maternal centrioles are heterogeneous in duplicating capacity, during meiosis only one centriole in each of the centrosomes of a meiosis-I spindle pole retains duplicating capacity, the reproductive centrioles are successively cast off into PBs, and finally a mature egg inheriting a nonreproductive centriole alone is formed, and the presence of a single reproductive centriole is sufficient condition for embryonic development in starfish.     

Kinetics and regulation of de novo centriole assembly. Implications for the mechanism of centriole duplication

BACKGROUND: Centriole duplication is a key step in the cell cycle whose mechanism is completely unknown. Why new centrioles always form next to preexisting ones is a fundamental question. The simplest model is that preexisting centrioles nucleate the assembly of new centrioles, and that although centrioles can in some cases form de novo without this nucleation, the de novo assembly mechanism should be too slow to compete with normal duplication in order to maintain fidelity of centriole duplication. RESULTS: We have measured the rate of de novo centriole assembly in vegetatively dividing cells that normally always contain centrioles. By using mutants of Chlamydomonas that are defective in centriole segregation, we obtained viable centrioleless cells that continue to divide, and find that within a single generation, 50% of these cells reacquire new centrioles by de novo assembly. This suggests that the rate of de novo assembly is approximately half the rate of templated duplication. A mutation in the VFL3 gene causes a complete loss of the templated assembly pathway without eliminating de novo assembly. A mutation in the centrin gene also reduced the rate of templated assembly. CONCLUSIONS: These results suggest that there are two pathways for centriole assembly, namely a templated pathway that requires preexisting centrioles to nucleate new centriole assembly, and a de novo assembly pathway that is normally turned off when centrioles are present.     

The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts.

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis.