A cell must build a bipolar mitotic spindle in order to faithfully segregate replicated DNA. To do so, multiple microtubule nucleation pathways are utilized to generate the robust spindle apparatus. TPX2, a microtubule binding protein, holds crucial roles in both the Ran-dependent and Augmin-dependent pathways where microtubules are nucleated near the chromosomes and from pre-existing microtubules. However, the exact role TPX2 plays in branching microtubules is less understood. Here, we explored the effect of truncating the essential TPX2 C-terminal 37 amino acids on Augmin localization and branching microtubule activity. First, we depleted LLC-Pk1 cells of the Augmin subunit HAUS6 and show that microtubule nucleation around the chromosomes following a nocodazole washout is strongly reduced leading to exaggerated kinetochore microtubule growth. Next, we depleted endogenous TPX2 in LLC-Pk1 cells harboring full length or truncated TPX2 bacterial artificial chromosome (BAC) DNA. Results show that TPX2 710 LAP cells have reduced Augmin localization on the spindle fibers, which correlates with reduced microtubule regrowth in the chromosomal region. In TPX2 710 LAP cells, regrowth was like Augmin depleted cells. Therefore, we provide evidence that the far C-terminus of TPX2 is required for branching microtubule nucleation and that kinetochore microtubule growth is Augmin-independent. In addition, we investigated cell cycle regulation of TPX2 by mutating the S738 phosphosite in the C-terminal motor interacting region. We utilized BAC recombineering to create phospho-mimetic and phospho-null mutants. In combination with plasmid DNA knockdown/rescue, overexpression and spindle assembly assays, we show that the phosphorylation of the C-terminal domain contributes to early mitotic events. LLC-Pk1 cells showed a significant increase in aberrant spindle morphology and reduced spindle stability in the presence of 738A and absence of endogenous TPX2. While rescue with the alanine mutant caused in an increase in multipolar spindles, overexpression resulted in a strong dominant negative monopolar phenotype. Therefore, S738 appears to contribute to mitotic force regulation during mitosis. In conclusion, the far C-terminus of TPX2 and its regulation play a role in the formation of a proper mitotic spindle.

Mitosis is the process by which new cells are formed. Following accurate duplication of chromosomes, a cell must segregate its chromosomes into separate daughter cells with great accuracy. Failure to do so can result in genomic instability. Thus, entry into mitosis is tightly regulated via complex protein interactions. Microtubules (MTs) are versatile Tubulin polymers that constitute a considerable portion of the cytoskeleton, and it is the dramatic rearrangement of MTs upon mitotic entry that is required to build the mitotic spindle – the structure responsible for segregating the duplicated sister chromatids. MTs are modulated by MT-Associated Proteins (MAPs) that enact major MT rearrangements during mitosis. Identifying and understanding the role of MAPs is essential to the study of MT behaviour during mitosis. Recently, an RNAi screen for Drosophila MAPs identified two proteins that are the subject of this thesis; Msd1 and dTD-60. This thesis demonstrates that Msd1 is a novel MAP that is a component of the recently identified Augmin complex – the action of which is to generate a novel and redundant population of MTs within the mitotic spindle from existing MTs. Experiments described below demonstrate that Msd1 is required for Augmin-directed MT generation, and further investigate the role of this novel population of MTs within the developing fruit fly. Furthermore, a role for Msd1 in central spindle formation during anaphase in Drosophila is identified. dTD-60, the Drosophila homologue of human TD-60 (hTD-60), is the subject of another study described in this thesis. While hTD-60 has a role in metaphase progression through interaction with the Chromosomal Passenger Complex, a contrasting role for dTD-60 is investigated here. This thesis describes both a divergent localisation and phenotype of dTD-60, and further investigates the role of dTD-60 and its interactors in mitotic spindle formation.

<p>Conserved across metazoans, cell division depends upon the synchronous assembly and disassembly of a robust, mitotic spindle for the congression and separation of duplicated chromosomes. Composed of mostly microtubules, mitotic spindle generation depends on three different microtubule nucleation mechanisms to build its distinctive bipolar assembly. These three mechanisms are centrosomal-based, kinetochore-based, and branching microtubule nucleation. Branching microtubule nucleation occurs when microtubules nucleate from the sides of pre-existing microtubules within the mitotic spindle. Without branching microtubules, a weaker spindle apparatus can result in mitotic delay, chromosomal misalignment, multi-polar spindles, and/or aneuploidy. </p> <p>Several important complexes and proteins mediate branching microtubule nucleation. These proteins are the γ-tubulin ring complex (γ–TuRC), the homologous to augmin subunits (HAUS) complex (or simply augmin), the targeting protein for Xklp2 (TPX2), colonic and hepatic tumor overexpressed gene (chTOG), and echinoderm microtubule-associated protein-like 3 (EML3) among others. This work focused on discerning the molecular architecture of the augmin complex while also endeavoring to establish heterologous expression and purification methodologies for the γ–TuRC and TPX2. </p> <p>Augmin consists of proteins HAUS1–8 (H1–8) which bind to the sides of pre-existing microtubules and orient the γ–TuRC, the template for making microtubules, via NEDD1 to create new microtubules at shallow angles (~<20°). Despite its importance in cell division, the structure of augmin has eluded determination. This work utilized a multi-pronged approach of the baculovirus insect cell protein complex expression, cryo-EM, new protein structure prediction methodologies, and crosslinking mass spectrometry (CLMS) to elucidate the molecular architecture of the augmin complex. Further work studying the isolation, structure prediction and comparison across model organisms, and phosphorylation studies was also conducted. The results will aid the structure-assisted development of novel chemotherapeutics that target the augmin complex as well as provide deeper insights into how this complex functions in cell division. </p> <p>To help better understand the molecular mechanisms, regulation, and interactions between the different machinery involved in branching microtubule nucleation, the γ–TuRC and TPX2 also became a focus of this work. My primary effort was to overexpress and purify from the heterologous baculovirus insect cell protein complex expression system sufficient quantities of γ–TuRC for biochemical and biophysical characterization. Thus, efforts shifted to establish an expression and purification methodology for this complex. Similarly, a methodology for purification of TPX2 were also initiated. The goal of these endeavors is to establish <em>in vitro</em> biochemical reconstitution of branching microtubule nucleation utilizing the augmin complex, γ–TuRC, and TPX2 utilizing total internal reflection fluorescence microscopy (TIRF-M). </p> <p>Lastly, in unrelated work, a section on other work focuses on the roles of anti-CRISPR proteins that inhibit the Csy surveillance complex from <em>Pseudomonas aeruginosa</em> can be found. Cryo-EM studies revealed the structures of AcrIF4, AcrIF7, and AcrIF14. These anti-CRISPR proteins inhibit the Csy complex by different mechanisms. AcrIF4 prevents conformational changes necessary to recruit a Cas2/3 nuclease for degradation of invading mobile genetic elements while AcrIF7 acts as a dsDNA mimic preventing invading phage DNA recognition. Lastly, AcrIF14 functions by binding in the grove where the crRNA of Csy is and prevents hybridization between target invading MGE DNA and the crRNA. These mechanisms exemplify convergent evolution among anti-CRISPR proteins while also showing the diversity of structures produced by phages in their ongoing molecular arms race with their hosts.</p>

La division cellulaire est essentielle pour la survie de chaque être vivant. Au cours de ce processus, les chromosomes de la cellule en division sont transmis aux deux cellules filles. La répartition des chromosomes est orchestrée par une structure cellulaire transitoire appelée fuseau mitotique (ou fuseau méiotique dans les cellules reproductrices). Le fuseau est composé de microtubules, de nombreuses protéines et de moteurs moléculaires, qui interagissent de manière complexe et précise aboutissant à l’organisation d’une structure bipolaire dynamique. Comme certains mécanismes moléculaires restent mal compris, nous avons choisi d'aborder la question de l'assemblage du fuseau méiotique dans des extraits d'oeufs de grenouille. Xenopus laevis est un organisme modèle car il est proche, d’un aspect phylogénétique, de l'homme, et il est particulièrement adapté à l’étude de la division cellulaire. Nous avons également utilisé une méthode in vitro (appelée spindle array ou puce à fuseaux) qui a été développée au sein du groupe de recherche auparavant, et qui offre certains avantages par rapport aux approches existantes. Une puce à fuseaux est composée de billes recouvertes de chromatine immobilisées selon des micro-motifs géométriques obtenus selon une technique d’impression par microcontact. L'assemblage des fuseaux méiotiques a été visualisé par microscopie confocale à fluorescence. Grâce à ces outils, nous avons, lors d’un premier projet, abordé le rôle de l’Augmin dans l'assemblage des fuseaux. L’Augmin est un complexe protéique récemment identifié grâce à son hypothétique rôle dans la nucléation de microtubules à partir de microtubules existants. Après déplétion de l’Augmin, nous avons constaté que la nucléation des microtubules était réduite et que les fuseaux avaient une morphologie anormale. De plus, ces derniers qui étaient essentiellement multipolaires sont progressivement devenus bipolaires grâce à une voie de nucléation des microtubules, découverte lors de notre étude, émanant des pôles acentrosomaux et qui est indépendante de l’Augmin. Nos résultats révèlent que l’Augmin est essentiel pour l’assemblage et la bipolarité du fuseau acentrosomal. Au cours d’un second projet, nous avons étudié les fonctions des chromokinésines kinésine-4 (Xklp1) et kinésine-10 (Xkid) dans l'assemblage des fuseaux et leurs mouvements. Xkid participe à la force d’éjection polaire nécessaire à la congression des chromosomes alors que Xklp1 contribue principalement à la régulation de la dynamique des microtubules. En étudiant l'assemblage de fuseaux dans des extraits après déplétion de Xkid, Xklp1 ou les deux, nous avons démontré que Xkid limite la dynamique des mouvements longitudinaux des fuseaux, contribue à la mise en place de la bipolarité et régule la longueur des fuseaux. Nous avons également quantifié la cinétique de nucléation des microtubules et confirmé le rôle de Xklp1 dans la régulation de la dynamique des microtubules. L’ensemble de nos travaux contribuent à une meilleure compréhension des mécanismes d’assemblage du fuseau méiotique et confirme la pertinence de notre méthode pour l'étude de sa morphogenèse. / Cell division is essential for the survival of every living organism. During this process, the chromosomes of the dividing cell are transmitted to the two daughter cells. The partition of the chromosomes is orchestrated by a transient sub-cellular structure called the mitotic spindle (or meiotic spindle in gamete cells). The spindle is composed of microtubules, numerous proteins and molecular motors, which interact in an intricate and yet precise manner leading to a highly dynamic and complexstructure. As some molecular mechanisms remain elusive, we have chosen to address the question of meiotic spindle assembly in Xenopus egg extracts. Xenopus laevis is a model system that is evolutionary close to human, and suitable for cell division studies. We have combined this with an in vitro assay - spindle array - which we developed prior to this work, and which provides advantages over existing approaches. A spindle array is composed of chromatin-coated beads that are immobilized according to geometrical patterns obtained by microcontact printing. The assembly of meiotic spindles wasvisualized by time-lapse fluorescence confocal microscopy. Using these tools, we first addressed the role of augmin in the assembly of meiotic spindles. Augmin is a recently identified protein complex that has been hypothesized to induce microtubule nucleation from the side of preexisting microtubules. By depleting augmin, we found that microtubule nucleationwas reduced and that spindles were morphologically impaired. Spindles were predominantly multipolar but finally reached bipolarity as a result of a newly uncovered augmin-independent microtubule nucleation pathway from acentrosomal poles. Our results thus reveal that augmin is essential for the proper establishment of the microtubule scaffolding and the bipolarity ofacentrosomal spindles. Secondly, we investigated the functions of the chromokinesins kinesin-4 (Xklp1) and kinesin-10 (Xkid)in acentrosomal spindle architecture and motions. Xkid plays a major role in the polar ejection forces leading chromosome movements during congression while the main function of XKlp1 is to regulate microtubule dynamics. We studied spindle assembly in depleted extracts and we report that Xkid limits the dynamics of spindle longitudinal movements, contributes to spindle bipolarity and affects spindle length while XKlp1 controls the spindle microtubule mass. Altogether these findings contribute to a better understanding of meiotic spindle assembly and confirm the pertinence of our method to study spindle morphogenesis.

Aurora A (AurA) is an important mitotic kinase mainly studied for its involvement in cell cycle progression, centrosome maturation, mitotic spindle pole organization and bipolar spindle formation. It localizes to duplicated centrosomes and spindle microtubules (MTs) during mitosis where it regulates various factors participating in metaphase spindle formation. AurA is degraded late in mitosis suggesting that it might also have a function in anaphase. In this study we focused in understanding AurA function during anaphase in two different experimental systems. First, we kept AurA active in cycled Xenopus egg extracts and found that MTs maintained their mitotic organization longer throughout mitotic exit. We also observed chromosome segregation defects and problematic nuclear envelope formation. These observations indicate that AurA activity needs to be down-regulated for the transition from metaphase back to interphase. To get insights into the role of AurA during metaphase-anaphase transition we initially asked whether its kinase activity is still necessary for the maintenance of the metaphase spindle. We saw that the inhibition of AurA kinase activity in metaphase resulted to a collapse of the established metaphase spindle in HeLa cells. Indicating that AurA activity is necessary for the metaphase spindle maintenance. Then, we looked whether AurA kinase activity is still necessary during anaphase. We inhibited AurA at the onset of anaphase in Hela cells and found that anaphase spindles were smaller. We also observed that the MT structure responsible for anaphase spindle elongation, the central spindle, was defectively assembled and organized. Moreover, in cells where AurA was inhibited segregation of chromosomes was defective. These results indicate that AurA kinase activity is necessary for anaphase spindle elongation, central spindle assembly and organization and chromosome segregation. To understand further how AurA regulates anaphase spindle formation we looked known AurA substrates. We depleted TACC3, a known AurA substrate involved in MT formation earlier in mitosis and observed that TACC3 depletion phenocopied AurA inhibition. This indicates that TACC3 has a function in MT organization and chromosome segregation during anaphase and this function could possibly be regulated by AurA. In this study we have demonstrated that AurA activity is essential for metaphase spindle maintenance. We also found that during anaphase when AurA is either maintained active or inhibited MT organization is greatly affected and chromosome segregation is defective. Suggesting that AurA activity needs to be tightly controlled during anaphase for a correct completion of mitosis. / Aurora A (AurA) es una quinasa mitótica importante que se ha estudiado principalmente en su papel durante la progresión del ciclo celular, la maduración del centrosoma, la organización y la formación del polo y del huso mitótico. Durante la mitosis, AurA se localiza en los centrosomas duplicados y en los microtúbulos (MTs) del huso y se ha observado que regula varios factores que participan en la formación del huso mitótico. AurA se degrada al final de la mitosis indicando que pueda tener una función durante la anafase. En este estudio nos hemos centrado en la comprensión de la función de AurA durante la anafase en dos sistemas experimentales diferentes. En primer lugar, utilizando extractos de huevos de Xenopus hemos mantenido AurA activa durante la transición de metafase a anafase y hemos visto que los MTs del huso mitótico mantienen su organización durante más tiempo. También hemos observado que cuando AurA se mantiene activa existen defectos en la segregación cromosómica y la formación de la membrana nuclear. Esto indica que la actividad de AurA tiene un papel regulador sobre los MTs y la chromatina durante la transición de la metafase a la interfase. Para entender cual es la función de AurA durante la transición de metafase a anafase primero hemos estudiado si la actividad de la quinasa es necesaria para el mantenimiento del huso mitótico. Hemos visto que la inhibición de la actividad quinasa AurA resultó en el colapso del huso durante la metafase en células HeLa. Esto indica que la actividad de AurA es necesaria para el mantenimiento del huso mitótico de metafase. A continuación hemos analizamos si la actividad quinasa de AurA sigue siendo necesaria para la anafase. Para ello hemos inhibido AurA en células Hela al inicio de la anafase. En estas condiciones los husos de la anafase son más pequeños y la estructura de los MTs responsable del alargamiento del huso mitótico durante la anafase, el huso central, se organiza defectuosamente. Además, se encontraron errores durante la segregación de los cromosomas. Estos resultados indican que la actividad quinasa de AurA es necesaria para el alargamiento del huso durante la anafase y la organización y segregación cromosómica. Para entender el mecanismo de la función de AurA durante la anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 , un sustrato conocido de AurA que participa en la formación de MTs en las fase iniciales de la mitosis hemos encontrado que su eliminación de células HeLa produce el mismo fenotipo que la inhibición de AurA. Esto indica que TACC3 tiene una función en la organización de MT y la segregación de cromosomas durante la anafase y que esta función podría estar regulada por la quinasa AurA. En este estudio hemos demostrado que la actividad quinasa de AurA es esencial para el mantenimiento del huso mitótico. También hemos encontrado que durante la anafase cuando la quinasa AurA se mantiene activa o se inhibe la organización de los MTs del huso mitótico se ve muy afectada y los cromosomas se segregan defectuosamente. Por tanto los resultados de este estudio indican que la actividad quinasa de AurA está estrechamente controlada durante la anafase para el correcto cumplimiento de la mitosis.