Centrosomes are major microtubule organizing centres that set up an internal microtubule (MT) network contributing to cell shape and to the formation of the mitotic spindle during cell division. Rearrangement of this MT array can be dictated by the centrosome and occurs during cell adhesion, polarization and migration. However, little is known about what regulates centrosome assembly and maintenance. beta1-integrins are common cell surface receptors and we show that beta1-integrin signalling is necessary for modulation of centrosome dynamics. In an attempt to identify the downstream components of beta1-integrin signalling involved, we also discovered that the activation of focal adhesion kinase or integrin linked kinase are not required in maintaining centrosome integrity. This would indicate that a non-canonical signalling beta1-integrin pathway might be involved in controlling centrosomal dynamics. This gives us greater insight into the mechanisms that control centrosomal stability and may lead to the better understanding of diseases like cancer and diseases, i.e. lissencephaly, which involve defects in cell polarization and asymmetric cell division, where the centrosome seems to have an important role.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Additional advisors: Vithal K. Ghanta, Denise R. Shaw, Stephen A. Watts, Bradley K. Yoder. Description based on contents viewed Feb. 20, 2009; title from PDF t.p. Includes bibliographical references.

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.

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This thesis project is focused on the study of the signaling module formed by the NIMA-related protein Nek6, Nek7, and Nek9 and their function during early mitosis, with particular interest in centrosome separation and maturation. Nek9/Nercc1 was identified by Dr. Joan Roig. Nek9 is expressed in all cell lines and tissues studied is inactive during interphase while during mitosis is activated through phosphorylation by Plk1 which is in fact able to bind Nek9 and subsequently phosphorylates Nek9 on its activation loop. During mitosis Nek6 and Nek7 bind the C-terminal of Nek9. Once active, Nek9 can phosphorylate Nek6 and Nek7, thus activating them. Active Nek9 localizes at centrosome, suggesting that Nek9/Nek6-7 has important functions in the organization of microtubules during cell division. Confirming this idea, it has been shown that the microinjection of anti-Nek9 module induces arrest in prometaphase with disorganized spindle structures and misaligned chromosomes, or leads to abnormal mitosis resulting in aneuploidy. In the same direction, interference with the function of Nek7 or Nek6 leads to abnormal mitotic progression and spindle formation. We described how the Nek9/Nek6-7 module could provide a link connecting Plk1 and Eg5 in the context of centrosome separation. we analyzed the effects of Plk1, Eg5, Nek9, Nek6 or Nek7 down-regulation by RNAi on the extent of separation of duplicated centrosomes in prophase cells and we observed how this downregulation was affecting centrosome separation. We determine whether the activation of Nek9 or Nek6 could induce centrosome separation trasfecting cells with the active form of these two kinases; a considerable amount of cells that were in interphase shown separate centrosome demonstrating that Nek9/Nek6 are sufficient to induce centrosome separation. To test whether active Nek9 and Nek6 exerted their effect through the regulation of Eg5 we simultaneously transfected the cells with Eg5 siRNAs and we completely lost the centrosome separation described above. We demonstrated by immunofluorescence that the key event during centrosome separation was the recruitment of Eg5 at centrosomes and that the down-regulation of Plk1, Nek6, Nek7 or Nek9 resulted in prophase cells with unseparated centrosomes because Eg5 was not properly recruited. To prove whether the phosphorylation on Ser-1033 controls the accumulation of Eg5 to centrosomes and centrosome separation during early mitosis we transfected cells with wild type Eg5 or Eg5 S1033A; the wild type form of the kinesin was able to localize at centrosome and rescue the normal phenotype while Eg5 S1033A was not able to localize and resulted in cells delayed in mitosis. Plk1, the Nek9 activator, is involved in the regulation of centrosome maturation during early mitosis. Centrosome maturation refers to the process through which centrosomes increase size and microtubule nucleation activity and requires the accumulation of γ-TuRC complexes at centrosome. This recruitment depends on Nedd1 that acts as γ-Tubulin targeting factor. Plk1 depletion prevents accumulation of Nedd1 at centrosome. Our experiments show the importance of Nek9 in the regulation of centrosome maturation downstream of Plk1. Depletion of Nek9 by siRNA determined a decrease of γ-Tubulin and Nedd1 at centrosome. Further we investigated the upstream role of Plk1 depleting Plk1 and trasfecting active Nek9 and it was able to rescue the normal phenotype. Nek9 can interact with Nedd1 during mitosis and phosphorylates it provoking its accumulation at centrosome. The no-phosphorylable form of Nedd1 was not able to accumulate at centrosome and support the accumulation of γ-Tubulin there, determining a delay of the cells in prometaphase. Our results show that Nek9 is the link between Plk1 activity and the recruitment of Nedd1 to the centrosome and that the pathway formed by Plk1/Nek9/Nedd1 can be a key element in the control of mitotic centrosome maturation.

Le transport subcellulaire et la traduction localisée des molécules d'ARNm semble être un processus très répandu et important pour contrôler la distribution asymétrique des protéines dans les cellules. L’ARNm, Bsg25D, connu pour se localiser aux centrosomes et aux microtubules astraux dans les embryons de drosophile au cours des premiers événements d'embryogenèse, a été sélectionné pour déterminer le rôle et l'importance du ciblage de l'ARNm à l'appareil mitotique lors de la division cellulaire. La localisation de Bsg25D aux centrosomes dans les embryons de drosophile est conservée entre espèces telles que D. melanogaster, D. simulans et D. yakuba. Bsg25D encode une protéine qui est étroitement liée à la Ninein (Nin) et à la Ninein-like protein (Nlp), deux protéines associées aux centrosomes présentes dans les cellules mammifères. L’analyse structure-fonction démontre que la région codante et la région 3’UTR de Bsg25D sont nécessaires pour son ciblage. Ceci suggère qu’un élément de régulation en cis, qui favorise sa localisation se situe dans la région codante + 3’UTR. / The subcellular transport and localized translation of mRNA molecules is emerging as a highly prevalent and important process for controlling asymmetric protein distribution in cells. A candidate mRNA, Bsg25D, known to localize to centrosomes and astral microtubules in Drosophila embryos during early events of embryogenesis, was selected to determine the role and importance of mRNA targeting to the mitotic apparatus during cell division. The localization of Bsg25D to centrosomes in Drosophila embryos is conserved between species such as D. melanogaster, D. simulans and D. yakuba. Bsg25D encodes a protein closely related to centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) in mammalian cells. Structure function analysis revealed that the coding and 3’UTR of Bsg25D are necessary for its targeting pattern, suggesting that a cis-regulatory motif that drives its localization, is in the coding + 3’ UTR region.

Primary cilia are nonmotile, hair-shaped organelles that extend from the basal body in the centrosome. The present study is the first investigation of this organelle in the oligodendrocyte lineage in vivo. I used immunohistochemical approaches in normal and cilia-deficient mutant mice to study cilia in relation to oligodendrogenesis and myelination. Primary cilia immunoreactive for Arl13b and ACIII were commonly present in NG2+ oligodendrocyte progenitor cells (OPCs), in which cilia-associated pathways control proliferation, differentiation, and migration. The loss of primary cilia is generally associated with enhanced Wnt/β-catenin signaling, and Wnt/β-catenin signaling has been shown to promote myelin gene expression. I examined whether the lack of cilia in the oligodendrocyte lineage is associated with elevated Wnt/β-catenin activity. I found that absence of a primary cilium was associated with with higher levels of TCF3, and with β-galactosidase in Axin2-lacZ Wnt reporter mice. This evidence supports the proposal that cilia loss in oligodendrocytes leads to enhanced Wnt/β-catenin activity, which promotes myelination. Cilia are dependent on the centrosome, which assembles microtubules for the cilium, the cytoskeleton, and the mitotic spindle. Centrosomes are the organizing center for microtubule assembly in OPCs, but this function is decentralized in oligodendrocytes. I found that the intensity of centrosomal pericentrin was reduced in oligodendrocytes relative to OPCs, and γ-tubulin was evident in centrosomes of OPCs but not in mature oligodendrocytes. These decreases in centrosomal proteins might contribute to functional differences between OPCs and oligodendrocytes. The importance of cilia in the oligodendrocyte lineage was examined in Tg737orpk mice, which have a hypomorphic IFT88 mutation resulting in decreased cilia numbers and lengths. These mice showed marked, differential decreases in numbers of oligodendrocytes and myelin, yet little or no change in OPC populations. It appears that sufficient cells were available for maturation, but lineage progression was stalled. There were no evident effects of the mutation on Wnt/β-catenin. Factors that might contribute to the abnormalities in the oligodendrocyte lineage of Tg737orpk mice include decreased cilia-dependent Shh mitogenic signaling and dysregulation in cilia-associated pathways such as Notch and Wnt/β-catenin.

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.