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.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). The current standard of care is the combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but this only results in a 60% over-all 5-year survival rate, thus highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics that is being clinically evaluated for combination therapy. Rational selection of companion therapeutics for HDACi is difficult due to their poorly understood, cell-type specific mechanisms of action. To understand these mechanisms better, we developed a pre-clinical model system of response to the HDACi belinostat. Using this model system, we identified two major responses. Resistance, consisting of a reversible G1 cell cycle arrest with little induction of apoptosis; or sensitivity, consisting of mitotic arrest and high levels of apoptosis. In this dissertation, we determine that the induction of G1 cell cycle arrest is due to the increased expression of cyclin dependent kinase inhibitors (CDKi) that bind to and inhibit the cyclin E/CDK2 complex thereby blocking the final repressive phosphorylation steps of Rb protein. Repression of transcriptional elongation blocked CDKi upregulation and prevented G1 cell cycle arrest in belinostat-resistant cells. Additionally, we identified that belinostat arrests sensitive cells prior to metaphase and belinostat-resistant cells slow-down in mitosis but complete the process prior to arresting in G1. The combination of belinostat with the microtubule-targeting agent, vincristine resulted in strong synergistic induction of apoptosis by targeting mitotic progression. Furthermore, this combination prevents polyploidy, a key mechanism of resistance to microtubule targeting agents. Finally, we utilized selective class one HDAC inhibitors to identify the individual contributions of HDACs in the eliciting the responses observed with belinostat treatment. HDAC1&2 inhibition recapitulated the belinostat-resistant phenotype of G1 cell cycle arrest with little apoptosis, in both belinostat-resistant and sensitive cell lines. HDAC3 inhibition resulted in the induction of DNA damage, increased S phase and the induction of apoptosis in belinostat-resistant cells. Belinostat-resistant cells did not have observable effects to HDAC3 inhibitor alone but when combined with vincristine had significantly increased G2/M population at early time points. This suggests that HDAC3 maintains roles in DNA replication and also in mitotic progression. HDAC3 inhibition combined with vincristine resulted in a significant increase in polyploidy, suggesting that HDAC3 might not regulate the expression of apoptotic regulating factors as belinostat does.

Aluminium toxicity is the main limiting factor in crop production on acid soils. The main symptom of aluminium toxicity is a rapid inhibition of root growth, but the mechanism of root growth cessation remains unclear. In this diploma thesis we deal with the question of whether phospholipases PLDα1 and PLDδ may play a role in the mechanism of aluminium toxicity. We compared the responses of plants lacking PLDα and PLDδ with WT plants. Growth analysis of roots was performed in hydroponic conditions. The most sensitive part of roots was transient zone in which cells were dying earlier. It was further found that pldα1 plants were less sensitive on aluminium toxicity because their roots showed less growth inhibition than WT. Pldδ plants did not differ from WT plants in their response to aluminum. During further analysis of the pldα1 reactions, it was found that the root cells were capable of cell expansion during aluminum toxicity, and the cellular malformations were formed on the roots. This phenomenon was associated with faster reorientation and even depolymerization of cortical microtubules in response to toxic aluminium in pldα plants compared to WT plants. The results indicated that PLDα1 molecule affects the stability of cortical microtubules. Microtubules were less stable and they depolymerized...

Les modifications post-traductionnelles des microtubules sont accumulées sur la queue carboxy-terminale des tubulines α et β, situées à l'extérieur des microtubules. Ces modifications peuvent réguler sélectivement les interactions avec les moteurs moléculaires et les protéines associées aux microtubules (MAPs). Ces interactions sont essentiels pour les fonctions cellulaires et demandent une régulation stricte. La glycylation est une modification que génère des chaînes latérales glycine sur les protéines. Jusqu'à présent, la glycylation a été à peine étudiée, et la plupart des travaux se sont concentrés sur son rôle potentiel dans les cils et les flagelles. Peu est connu sur le rôle de la glycylation des microtubules dans les cils primaires. Les cils primaires sont des organelles sensorielles, impliquées dans la transduction des signaux et dans la progression du cycle cellulaire. Récemment, une étude approfondie de 13 023 gènes dans le cancer colorectal a révélé que les tumeurs individuelles accumulent une moyenne d'environ 90 gènes mutés. Un de ces gènes potentiels de cancer est la glycylase TTLL3. Notre équipe a testé les deux mutations décrites pour TTLL3 et a pu constaté que chacun d'entre eux conduit à une perte complète de l'activité de cette glycylase in vivo et in vitro. Mon travail vise donc à élucider le rôle de glycylation de protéines dans la signalisation cellulaire et ses conséquences pour la formation du cancer du côlon. J'ai analysé des échantillons provenant de patients par RT-PCR quantitative et j'ai trouvé une diminution des niveaux d'expression de TTLL3 dans les cancers. Les souris TTLL3-knockout ont été soumises à un modèle murin de carcinome du côlon, induit chimiquement à la base de l'azoxyméthane (AOM) et du sulfate de dextran de sodium (DSS). Mes données montrent une formation tumorale élevée dans le groupe TTLL3-KO, ce qui suggère que la perte de la glycylation est liée au développement du cancer du côlon. La glycylation se trouve dans les cils primaires, et les défauts ciliaires ont été décrits dans différents types de tumeurs solides. La présence de cils primaires et l'importance de la glycylation dans le côlon n'étaient pas encore connues au début de mes travaux. Collectivement, mes résultats indiquent que la glycylation est nécessaire, mais pas indispensable pour les cils primaires. Remarquablement, j'ai pu démontrer la présence de cils primaires sur les cellules épithéliales du côlon pour la première fois, et j'ai mis en évidence un défet de ces cils dans les souris TTLL3-KO in vitro et in vivo. Par ailleurs, j'ai démontré que le dysfonctionnement des cils coliques dans les souris KO TTLL3 est associé à une augmentation de l'activité proliférative des cellules épithéliales. Par conséquent, la glycylation pourrait être importante pour la genèse et le fonctionnement des cils primaires. Dans le côlon, l'absence de la glycylase TTLL3 peut entraîner un manque de la glycylation qui favorise la formation de tumeurs. / Tubulin posttranslational modifications are involved in the regulation of many microtubule functions. Glycylation has been related to the stability and maintenance of motile cilia in different organisms including mammals. We had previously shown that some colon-cancer related mutations in the glycylating enzyme TTLL3 lead to a complete loss of enzymatic activity, which brought up a surprising link between this rather cilia-specific tubulin modification and cancer. To evaluate potential role of glycylation in colon carcinoma formation we first confirmed the link between TTLL3 and colon cancer in a greater cohort of patients. We next studied TTLL3-knockout mice, which strikingly did not show any obvious phenotypic alterations or spontaneous cancer development. However, when submitted to a murine model of chemically induced colon carcinoma, TTLL3-knockout mice show a higher level of tumor formation, pointing towards an acceleration of colon cancer development. Because glycylation of microtubules has been specifically detected on ciliary tubulin, we next analysed the presence of primary cilia in colon epithelium. While in most organs and tissues a second glycylating enzyme, TTLL8, is expressed, TTLL3 is the unique enzyme in colon. We found a significantly reduced number of primary cilia in TTLL3-KO colon epithelium, suggesting that similar to motile cilia, primary cilia are maintained by glycylation of the axonemal tubulin. Moreover, we measured a strongly increased mitotic index in colon epithelial cells isolated from TTLL3-KO mice, indicating that his loss of cilia is accompanied by decreased level of cell cycle control. Thus we have demonstrated for the first time a tight link between the posttranslational glycylation of the microtubule cytoskeleton, the control of cell cycle and the acceleration of cancer development.

No description available.

Oocytes of most species assemble and maintain a functional bipolar spindle in the absence of centrosomes. Strikingly, after bipolar spindle formation, oocytes arrest in metaphase for several hours before fertilisation. How the dynamic spindle maintains its bipolarity during this long arrest is poorly understood. I hypothesise that the bipolar spindle is stably maintained by changes in the distribution of microtubule-associated proteins (MAPs) on the spindle during the long oocyte arrest. To test this, I generated transgenic flies expressing GFP-tagged microtubule-associated proteins (MAPs), and found that 13 out of 24 proteins change localisation between early and late oocytes. I refer to these changes in MAP localisation after establishment of bipolarity as 'spindle maturation'. In order to identify the molecular mechanisms triggering MAP relocalisation, I manipulated the kinase activity of the cell cycle regulator Cdk1 by over-expressing non-degradable cyclin A or B, the major activators of Cdk1. Their expression prevented re-localisation of distinct sets of MAPs, and disrupted spindle bipolarity and accurate chromosome segregation in oocytes. Kinesin-6 Pavarotti/MKlp1 localised strongly to the spindle equator in late oocytes, whilst nearly always absent from this region in early oocytes. The localisation of Pavarotti to the spindle equator in late oocytes was reduced when cyclin B is over-expressed in oocytes, suggesting a role for Cdk1/cyclin B complex in regulating Pavarotti localisation. Indeed, a Pavarotti/Mklp1 mutant non-phosphorylatable by Cdk1 prematurely localised to the meiotic spindle and disrupted spindle bipolarity. Moreover, removal of Pavarotti from the metaphase-I spindle by RNAi induced spindle defects in oocytes. Therefore, it is likely that the microtubule cross-linking activity of Pavarotti enhances the stability of the metaphase-I spindle during the long arrest. Consistent with this, I found that the microtubule density in the spindle equator is higher in late oocytes. Altogether, I propose that remodelling the molecular architecture of the spindle during the long oocyte arrest is important to stabilise the bipolar spindle without centrosomes.

Chloroplasts are organelles that convert light energy to chemical energy through photosynthesis. The movement of chloroplasts within the cell for the optimization of light absorption is crucial for plant survival. Cellular motor proteins and cytoskeletal tracks can facilitate transport of organelles. As an ancient superfamily of microtubule-dependent motors, kinesins participate in various cellular activities including cytokinesis, vesicle and organelle movements. Based on phylogenetic relationships and functional analysis, the kinesin superfamily has been subdivided into more than 14 families, most of which can be found in plants. With the ever increasing amount of genomic information, it is important and beneficial to systematically characterize and document kinesins within an organism. As a result of my collaborative work with other members of the Vidali lab, a detailed phylogenetic characterization of the 76 kinesins of the kinesin superfamily in the moss Physcomitrella patens is reported here. We found a remarkable conservation of families and subfamily classes with Arabidopsis, which is important for future comparative analyses of functions. Some of the families are composed of fewer members, while other families are greatly expanded in moss. To improve the comparison between species, and to simplify communication between research groups, we proposed a classification of subfamilies based on our phylogenetic analysis. As part of my efforts in studying chloroplasts motility, I investigated the function of two members of Physcomitrella kinesin family 14 class V proteins, Ppkin14-Va and -Vb. These two proteins are orthologs of the Arabidopsis KAC proteins which mediate actin-based chloroplast movement in Arabidopsis thaliana. In contrast, in the Physcomitrella both actin filaments (AFs) and microtubules (MTs) participate in chloroplast movement. Our results show that Ppkin14-Vs are important for maintaining chloroplast dispersion. They also function during chloroplast light avoidance responses via an AF-dependent, rather than MT-dependent mechanism. Although two Ppkin14- Vs do not act as MT-based motors, our phylogenetic study on moss kinesins provides an important source of information to track other potential kinesins that are predicted to move chloroplasts on MTs.

Or Man Wai. / "January 2002." / "glued" in title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.

Le terme de CLIP-170 désigne la protéine de lien cytoplasmique de 170 KDa, isolée par Rickard et Kreis (1990). In vitro, elle constitue un lien statique entre les endosomes et les microtubules (MTs). En chacun des points où la CLIP-170 est localisée, elle se co-distribue avec le complexe moteur dynéine/dynactine (D/D). Les auteurs ont initialement établi un modèle de fonctionnement de concert de ces trois protagonistes : la CLIP-170 établirait le lien initial entre le cargo et le MT. Le complexe D/D serait recruté sur le cargo. Une fois le moteur associé au MT, le lien statique serait levé, rendant possible le mouvement. Ce modèle offrait une explication aux données d'immunolocalisation. Toutefois, le fonctionnement de concert de la CLIP-170 et du moteur moléculaire nécessitait que l'on puisse trouver une interaction entre les protagonistes. Les travaux présentés dans cette thèse apportent pour la première fois la preuve d'une interaction indirecte entre le complexe D/D et la CLIP-170. LIS1 est une protéine codée par le gène causal du syndrome de Miller-Dieker, une forme de lissencéphalie de type I. Elle sert d'adaptateur entre le domaine carboxy-terminal de la CLIP-170 et le complexe moteur dont elle régule l'activité. Nos résultats établissent que le domaine d'interaction de la CLIP-170 avec LIS1 est requis pour l'adressage de la première aux kinétochores prémétaphasiques. Il est nécessaire à l'adressage de LIS1 (et du complexe moteur) aux bouts (+) des MTs interphasiques. Nous présentons deux nouvelles approches permettant d'interférer avec le fonctionnement de la CLIP-170 tant en interphase qu'en mitose : la microinjection d'anticorps dirigés contre les domaines extrêmes de la protéine, ainsi que l'utilisation de siRNA dirigés contre l'ARNm la codant. Combinées aux techniques de suivi de protéines fluorescentes par vidéomicroscopie 3D développées au laboratoire, elles permettront d'interférer avec le fonctionnement de la CLIP-170 et de définir ainsi son rôle.