Polo-like kinase 4 (Plk4) has been identified as a molecular marker of resistance to therapy in cancer. Our laboratory has recently shown a motility defect in Plk4+/- murine embryonic fibroblasts (MEFs) compared to Plk4+/+. I hypothesized that Plk4 augments cancer cell motility. Plk4 depletion with siRNA in Plk4+/+ MEFs and HeLa cells suppressed invasion compared to control. Transient over-expression of Flag-Plk4 in cancer cell lines did not consistently increase invasion. However, modest Plk4 over-expression using stable clones showed higher invasion rates than non-induced. The RhoGEF Ect2, an important Plk4 substrate, transiently localized to protrusions in MEFs, suggesting a RhoA-based signalling cascade in motility. The effect of Plk4 heterozygosity on metastasis was tested in a transgenic mouse model but there was no significant difference in developing metastasis compared to wild type. Further studies are required to characterize the effect of Plk4 on motility, and its potential as a therapeutic cancer target.

Polo-like kinase 4 (Plk4) has been identified as a molecular marker of resistance to therapy in cancer. Our laboratory has recently shown a motility defect in Plk4+/- murine embryonic fibroblasts (MEFs) compared to Plk4+/+. I hypothesized that Plk4 augments cancer cell motility. Plk4 depletion with siRNA in Plk4+/+ MEFs and HeLa cells suppressed invasion compared to control. Transient over-expression of Flag-Plk4 in cancer cell lines did not consistently increase invasion. However, modest Plk4 over-expression using stable clones showed higher invasion rates than non-induced. The RhoGEF Ect2, an important Plk4 substrate, transiently localized to protrusions in MEFs, suggesting a RhoA-based signalling cascade in motility. The effect of Plk4 heterozygosity on metastasis was tested in a transgenic mouse model but there was no significant difference in developing metastasis compared to wild type. Further studies are required to characterize the effect of Plk4 on motility, and its potential as a therapeutic cancer target.

Chromosomal Instability (CIN) occurs in over 90% of all sporadic tumors and manifests as whole chromosome loss or gain, gene deletions, amplifications, inversion, and translocations. CIN is not only a hallmark of cancer but promotes tumorigenesis. CIN is caused by errors during mitosis and one major CIN-promoting mechanism is centrosome over-duplication (amplification); another cancer hallmark. Centrosome amplification causes abnormal mitotic spindle assembly, directly promoting chromosome mis-segration with consequent aneuploidy and other forms of CIN. Central to controlling centrosome numbers and function are the Polo kinases, including Polo-like kinase 4 (Plk4). Plk4 is a component of centrosomes and recognized as the master-regulator of centrosome function and duplication. Plk4 is a mitotic kinase whose levels increase throughout S-phase and G2 to peak in mitosis. During late mitosis, Plk4 localizes to a spot on parent centrioles, licensing this single site for future daughter centriole assembly. Plk4 activity initiates the hierarchial recruiment of two conserved essential centriole proteins: Ana2, followed by the cartwheel protein Sas6. By analysis in a yeast-2-hybrid screen, we identified several novel interactions of centriole proteins, including the interaction of Ana2 and Plk4. Plk4 phosphorylates Ana2 to both positively and negatively regulate centriole duplication. Our preliminary data suggests that Plk4 recruits Ana2 by phosphorylating a protein on the outer centriole surface, generating a phospho-landing platform, and that this Plk4 target is Sas4 (CPAP in humans). Notably, the Sas4 pattern on centrioles is complex, forming both a ring and an asymmetric spot during mitotic progression. Like Sas4, Ana2 is a Plk4 substrate, and when mixed with purified Ana2, Sas4 stimulates Ana2 hyperphosphorylation in vitro. Thus, Plk4 influences centriole assembly on multiple platforms.

Les centrosomes sont les principaux centres organisateurs des microtubules dans les cellules animales, impliqués dans la division, la motilité, la polarité cellulaire. Ils participent à l'élaboration du fuseau mitotique, qui permet la séparation des chromosomes dans les cellules filles. Dans les neuroblastes de drosophile en interphase, un des deux centrosomes maintient son activité et sa position apicale dans la cellule, alors que l'autre est inactivé et se déplace vers le pôle basal. La duplication des centrioles est initiée par la kinase Plk4 une seule fois par cycle cellulaire. Toute dérégulation des niveaux de Plk4 conduit à un défaut du nombre de centrosomes, à l'origine de pathologies comme le cancer et la microcéphalie. Pendant ma thèse, j'ai étudié les rôles et régulations de Plk4 in vivo dans les neuroblastes de drosophile. J'ai montré un nouveau rôle de Plk4 dans l'établissement de l'asymétrie des centrosomes durant l'interphase. Plk4 favorise un comportement basal des centrosomes en inhibant la nucléation des microtubules et l'ancrage au pôle apical. Plk4 régule négativement la localisation du co-activateur de l'APC/C, Fizzy-related, que j'ai identifié comme un régulateur positif de l'activation du centrosome. APC/C est une E3 ubiquitine-ligase, qui cible les protéines régulant le cycle cellulaire vers la dégradation. J'ai montré que Plk4 interagit avec ce complexe in vivo. Des mutations du motif de liaison à l'APC/C conduisent à la stabilisation de Plk4 et à une dérégulation de son accumulation au centrosome au début de l'interphase. Mon étude a donc démontré que dans les neuroblastes Plk4 coordonne la duplication des centrioles et le cycle des centrosomes. / The centrosome is the main microtubule-organizing centre of animal cells with important roles in cell division, motility and polarity. In cycling cells, upon duplication, two centrosomes form the mitotic spindle, the apparatus that physically segregates the chromosomes into the daughter cells. In Drosophila neural stem cells of the larval brain, called neuroblasts, during interphase, one centrosome stays active and static at the apical side of the cell, while the other one is inactive and moves toward the basal side of the cell. Centriole duplication, which occurs only once per cell cycle, is initiated by the Polo-like kinase 4 (Plk4). Deregulation of Plk4 levels leads to alteration in centrosome number, a defect that can cause diseases such as cancer and microcephaly. During my PhD I studied the role/s and regulation of Plk4 in vivo in Drosophila neuroblasts. I found that Plk4 plays an important role in establishing centrosome asymmetry during interphase. Plk4 promotes centrosome basal-like behaviour, through inhibition of MT nucleation and centrosome apical anchorage. Plk4 negatively regulates the centrosomal localization of the APC/C co-activator Fizzy-related (Fzr) that I identified as a positive regulator of centrosome activation. The APC/C complex is an E3 ubiquitin-ligase that targets cell-cycle-related proteins to degradation. I showed that APC/C and Plk4 interact in vivo. Mutations in the APC/C binding motif lead to stabilization of Plk4 that presents unscheduled accumulation at the centrosome in early interphase neuroblasts.In conclusion, my study demonstrates that in neuroblasts, the kinase Plk4 couples centriole duplication and centrosome cycles.

La méiose chez les femelles est un processus de réduction du génome permettant la formation d'un gamète haploïde, l'ovocyte. Dans la plupart des espèces animales, l'ovogénèse est associée à une élimination des centrioles, composants des centrosomes, qui sont les centres organisateurs des microtubules (MTOCs) canoniques. En l'absence de centrioles, le fuseau méiotique est assemblé grâce à la large contribution des MTOCs. Cependant, leur origine, l'importance de leur organisation et de la régulation de leur activité sont peu comprises. J'ai démontré que les MTOCs sont déjà présents dans les ovocytes précurseurs et sont progressivement rassemblés sur l'enveloppe nucléaire chez les ovocytes compétents pour les divisions méiotique. A l'entrée en méiose, les MTOCs sont distribués autour des chromosomes qui se condensent de façon strictement régulée au cours du temps. La perturbation de l'organisation des MTOCs par une surexpression de la kinase Polo-like 4 (Plk4) altère l'activité de nucléation des microtubules à l'entrée en méiose. L'activité anormale de ces MTOCs conduit à la formation de fuseaux non fonctionnels et à l'arrêt méiotique. / Female meiosis is a fundamental process of genome reduction leading to formation of a haploid gamete, the oocyte. In most animal species, oogenesis is associated with elimination of centrioles, constituents of centrosomes, which are canonical Microtubule Organizing Centers (MTOCs). In the absence of centrioles, meiotic spindle is assembled with a large contribution of acentriolar MTOCs. However, their origins, importance of organization and regulation of activity are poorly understood. I have shown that MTOCs pre-exist in the oocyte precursors and that they are progressively gathered on the nuclear envelope as oocytes gain competency for meiotic divisions. At entry into meiosis, MTOCs are redistributed around the condensing chromosomes in a strictly regulated timely fashion. Perturbation of such MTOC organization by transgenic overexpression of Polo-like kinase 4 (Plk4) leads to altered MT-nucleating activity at meiotic entry. Such abnormally active MTOCs mediate the formation of large and non-functional spindles and meiotic arrest.

No description available.

Centrosomes are organelles that promote microtubule growth. Normally, a single centrosome duplicates once each cell cycle to guide assembly of a bipolar mitotic spindle, ensuring that each daughter cell inherits an equal complement of the genome and a single centrosome. Centrosomes are composed of a pair of ‘mother-daughter’ centrioles and, during duplication, each mother centriole assembles one daughter at a single site. However, mother centrioles can inappropriately assemble multiple daughters, thereby generating centriole amplification (or overduplication), resulting in multipolar spindle assembly and, consequently, chromosome missegration - a driving force for chromosomal instability/aneuploidy which induces birth defects, miscarriage, and tumorigenesis. We have elucidated how the cell cycle control program regulates the centriole duplication machinery to limit centriole duplication to one event per cell cycle via the cell cycle-dependent regulation of Ana2/STIL and PLK4 degradation. In the case of the centrosome licensing factor Plk4, we found that autophosphorylation promotes its own destruction during interphase, which is then counteracted by the Protein Phosphatase 2A (PP2A) in complex with its Twins (tws) regulatory subunit during mitosis. This promotes stabilization of Plk4 and thus allows for the licensing of the mother centriole, making it competent to duplicate during the proceeding S-phase. While PP2Atws plays a positive role in regulating Plk4 to promote centriole duplication, we found that PP2A complexed with the Well-rounded (wrd) and Widerborst (wdb) regulatory subunits negatively regulates Ana2 by promoting its degradation to limit centriole duplication. PP2Awrd/wdb dephosphorylates numerous serine/threonine residues residing in Ana2, including several CDK phosphorylation consensus motifs. We found that CDK1/cycA and CDK2/cycE phosphorylate these residues to promote Ana2 stabilization from S-phase, the start of centriole duplication, to M-phase, the start of centriole duplication licensing. Interestingly, we found that the tumorigenic SV40 virus protein Small Tumor Antigen (ST) amplifies centrioles by targeting the PP2A complex to stabilize Plk4 as well as Ana2, underscoring the oncogenic importance of these newly discovered centriole duplication pathways. Finally, we shed insight into the mechanism for centriole amplification upon Ana2 stabilization by showing that Ana2 associates with Plk4 to promote Plk4 kinase activity as well as Plk4 stabilization.

No description available.

No description available.