PLK4 trans-Autoactivation Controls Centriole Biogenesis in Space

The deposited article is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.The deposited article version contains attached the supplementary materials within the pdf.Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions.Fundação Portuguesa para a Ciência e Tecnologia grants: (SFRH/BPD/87479/2012, PTDC/BBB-BEP/1724/2012, HMSP-CT/SAU-ICT/0075/2009, PTDC/SAU-OBD/105616/2008, EXPL/BIM-ONC/0830/2013, PTDC/BBB-BEP/1724/2012); EMBO installation grant; ERC starting grant: (PFE-GI-UE-ERC-2010-StG-261344).info:eu-repo/semantics/publishedVersio

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis

The deposited article is a post-print version (author's manuscript from PMC and available in PMC 2017 May 9).This publication hasn't any creative commons license associated.This deposit is composed by the main article and the supplementary materials are present in the publisher's page in the following link: https://www.sciencedirect.com/science/article/pii/S0960982216303001?via%3Dihub#sec4Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.ERC grant: (ERC-2010-StG-261344); FCT grants: (FCT Investigator, EXPL/BIM-ONC/0830/2013, PTDC/SAU-BD/105616/2008); EMBO installation grant.info:eu-repo/semantics/publishedVersio

Regulation of the tumor suppressor : the F-box Fbw7

Le système ubiquitine-protéasome joue un rôle central dans le contrôle de la progression du cycle cellulaire par la dégradation régulée de nombreuses protéines. Dans ce système, Fbw7 (aussi appelée Fbxw7, hCdc4, hAgo, Sel-10), est l'une des protéines F-box qui sert d'adaptateur de substrats pour l'une des plus importantes familles d'ubiquitine ligases : les complexes SCF (Skp1/Cullin/ F-box). Fbw7 assure la dégradation de plusieurs régulateurs positifs du cycle cellulaire : la cycline E, cMyc, c-Jun, Notch, Aurora A, mTOR, MCL1. En conséquence, l'altération des fonctions de Fbw7 conduit à des défauts de prolifération cellulaire, de différenciation et à de l'instabilité génomique. La mutation de Fbw7 dans les cancers entraîne une dérégulation de l'expression périodique cycline E qui n'est alors plus restreinte à la transition G1/S du cycle cellulaire. Nos résultats montrent qu'une isoforme, Fbw7, est exprimée dans les œufs de xénope matures arrêtés en métaphase II mais n'est pas fonctionnelle, expliquant la présence de grande quantité de cycline E dans les œufs à cette phase mitotique. Nous montrons que Fbw7 est maintenue inactive sous forme poly-ubiquitylée suite à sa phosphorylation par une PKC jusqu'à la fin des cycles embryonnaires rapides, au moment où la cycline E est brutalement dégradée. Nous montrons que la régulation négative de Fbw7 par PKC est conservée au cours des cycles cellulaires somatiques des cellules humaines, et contribue à l'expression périodique de la cycline E. Ces résultats mettent en évidence un nouveau mécanisme critique pour la régulation de Fbw7 au cours du cycle cellulaire et suggèrent que les fonctions de Fbw7 peuvent être altérées par une dérégulation de PKC, un phénomène observé dans de nombreux types de tumeurs humaines.The ubiquitin-proteasome system plays a central role in the control of cell cycle progression through the regulated degradation of numerous critical proteins. In this process, one key family of ubiquitin ligases are the SCF (Skp1/Cul-1/F-box) complexes, in which F-box-bearing proteins act as substrate-recruiting factors. Fbw7 (also known as Fbxw7, hCdc4, hAgo, Sel-10) is one such F-box protein. It controls the stability and thus the levels of several positive regulators of the cell cycle, including cyclin E, cMyc, c-Jun, Notch, Aurora A, mTOR, Mcl1. As a consequence of its biological roles, alterations of the functions of Fbw7 lead to defects in cellular proliferation, differentiation and genetic instability. As seen in cancers, mutation of Fbw7 leads to deregulation of cyclin E expression, which is no more restricted to the G1-S phase boundary of the cell cycle. Here we report that Fbw7, although expressed in mature Xenopus eggs arrested in metaphase II, is not functional, explaining why cyclin E can be stockpiled in this mitotic-like phase. We found that, in these eggs as well as in early Xenopus embryos, Fbw7 is maintained under a PKC-dependent poly-ubiquitylated state until the end of the early rapid cleavage cycles where cyclin E is abruptly degraded. Importantly, we show that this PKC-dependent negative regulation of Fbw7 is conserved during human somatic cell cycles, resulting into the periodic expression of cyclin E. These findings reveal a novel mechanism critical for the temporal regulation of Fbw7 and suggest that the key functions of Fbw7 can be altered by PKC dysregulation, a mechanism known to occur in many types of human tumours

General analytical models characterizing MBOC modulated signal

International audienc

Pericentrin-mediated SAS-6 recruitment promotes centriole assembly

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.info:eu-repo/semantics/publishedVersio

PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation

The deposited article version is the Epub Ahead of Print version of the article (the "Accepted Manuscript"), posted online 20th September 2018, provided by Company of Biologists. It has peer-review.The deposited article version contains attached the supplementary materials within the pdf.The centrosome is an important microtubule-organizing centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical to recruit several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation (Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α/β-tubulin. In Xenopus extracts, PLK4 assemblies additionally recruit PLK4's substrate, STIL, and the microtubule nucleator, γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similarly to centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and α/β-tubulin, leading to MTOC formation.We are thankful to Anna Akhmanova, Raquel Oliveira and Jeffrey B.Woodruff for reading and discussing the manuscript. We are also thankful to Catarina Nabais for the GFP control construct and Vladimir Joukov for the Xenopus Cep192 antibody. S.M.G was funded by an EMBO Long term fellowship ALTF 1088-2009, a Marie curie Intra-European fellowship (#253373) and a FCT postdoctoral fellowship. The collaboration with J.L. laboratory in the USA was supported by a The Company of Biologists travel grant. S.Z is funded by ERC grant ERC-COG-683258. Research in JL lab was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. M.B-D. Laboratory is supported by an ERC grant ERC-COG-683258 and FCT Investigator to MBD.info:eu-repo/semantics/acceptedVersio

The stability of Fbw7α in M-phase requires its phosphorylation by PKC

<div><p>Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCF^Fbw7 ubiquitin ligase. In human cancers bearing <i>FBXW7</i>-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in <i>Xenopus</i> eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.</p></div

Endogenous and ectopic Fbw7α are phosphorylated at Ser18 in M-phase.

<p>A. FLAG-Fbw7α was transfected or not in HeLa cells, as indicated. Total extracts of asynchronous cells (AS), cells synchronized in G1/S or arrested in pro-metaphase and recovered by shake-off (M) were analyzed by SDS-PAGE and immunoprobed for pS18-Fbw7α and FLAG-Fbw7α. The migration of Cdc27 was used as a marker of M phase (Cdc27^P), and anti-tubulin as a loading control. B. HeLa cell extracts (500 μg) from cells arrested in prometaphase were subjected to immunoprecipitation with anti-Fbw7 or control antibodies and analyzed by immunoblotting to detect phosphorylated Ser18 (pS18). Input 10% (i), supernatant after IP (s). C. HeLa cells grown on coverslips were co-transfected with FLAG-Fbw7α and either GFP- PKCα, PKCβ1, PKCε or PKCδ, as indicated. Coverslips were fixed and stained with FLAG antibodies and DAPI. Scale bar, 10 μm. D. HeLa cells were co-transfected with FLAG-Fbw7α and the different GFP-PKC constructs, as indicated. At 24 hr post-tranfection, cells were harvested and the levels of GFP-PKC, pS18-Fbw7α, FLAG-Fbw7α and tubulin were monitored by immunoblotting.</p

Fbw7 dimerization and cyclin E binding is reduced by a negative charge at Ser18.

<p>A. FLAG-and HA-tagged versions of Fbw7α either S18A or S18E were co-transfected in HeLa cells and analyzed for their interaction by immunoprecipitation with HA antibodies. Co-precipitated FLAG-tagged Fbw7α was detected by immunoblotting with an anti-FLAG antibody. The membrane was re-probed with HA antibodies. B. Fbw7 or control immunoprecipitates from HeLa cells transfected or not (-) with FLAG-Fbw7α-S18A or –S18E were mixed with <i>in vitro</i> translated [³⁵S]-Fbw7α-18A or -18E (with no tag in N-terminal), as indicated. Complexes were analyzed by phosphorimaging and immunoblotting. C. <i>In vitro</i> translated [³⁵S]-xCyclin E was first incubated with a recombinant Cdc25B phosphatase to activate its associated Cdk2 partner from the reticulocyte lysate and further incubated in MII-egg extracts with phosphatase inhibitors for 30 min. Phosphorylated cyclin E was mixed with FLAG-hFbw7α-18A or-18E immunoprecipitates. Complexes were analyzed by phosphorimaging and immunoblotting. D. <i>In vitro</i> translated [³⁵S]-FLAG-hFbw7α-465A, -18A or -wt were incubated in MII-egg extracts with phosphatase inhibitors for 60 min and mixed with GST-cyclin E bound to magnetic beads. The mutant hFbw7α-465A that cannot bind cyclin E serves as a negative control. Input represents 10% of the total extract (i), total beads (B). Complexes were analyzed by phosphorimaging and immunoblotting.</p

xFbw7α is phosphorylated by PKC on S18.

<p>A. <i>In vitro</i> translated [³⁵S]-xFbw7α was incubated in MII-egg extracts prepared with phosphatase inhibitors (left panel). [³⁵S]-xFbw7α-wt was incubated 1 hr in MII-egg extracts prepared with phosphatase inhibitors and an equal amount of sample was subsequently incubated with an excess of lambda protein phosphatase (λP) and analyzed by phosphorimaging (right panel). (B) <i>In vitro</i> translated [³⁵S]-xFbw7α-wt, -S18A, -S119A or -129A were incubated 1 hr in MII-egg extracts. C. Sequence alignment of the Fbw7α N-terminal region from <i>Xenopus</i> and human. D. GST-xFbw7α-wt or -S18A bound to magnetic beads were incubated with PKCα, activated (+) or not (-) and then submitted to immunoblot analysis with anti-pS18 antibodies. Total GST-Fbw7 was detected by Ponceau S staining. (E) GST-xFbw7α wt or S18A were incubated in MII-egg extracts plus phosphatase inhibitors, supplemented (+) or not (-) with Gö6976 and with [γ-³³P]-ATP for 1 hr at 23°C and analyzed by immunoblotting and by phosphorimaging for quantification of ³³P incorporation (lower panel).</p