Thesis (M.BmedSc.)--Victoria University of Wellington, 2010. / Includes bibliographical references.

Thèse (M.Sc.) -- Université Laval, 1997. / Bibliogr.: f. [83]-90. Publ. aussi en version électronique.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 128-149). Also available in electronic version.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 90-110). Also available on the World Wide Web.

Le cytosquelette de microtubule (MT) est essentiel dans de nombreux processus cellulaire. Il est notamment impliqué dans le trafic intracellulaire, la division cellulaire, la modification et le maintien de la forme de la cellule. En fonction du type cellulaire ou de son état de différenciation, les réseaux de MTs vont adopter des architectures différentes. Ces organisations sont définies par des contraintes géométriques intracellulaires et l'activité moléculaire de nombreuses protéines associées aux MTs (MAPs). Parmi ces protéines, des membres de la famille des MAP65s ont été identifiés. In vitro, elles forment des ponts entre les MTs pour les organiser en faisceaux. Le but de mon travail de thèse a été d'étudier in vitro le rôle de MAP65s dans l'auto-organisation d'un réseau de faisceaux de MTs. Dans un premier temps, j'ai mis au point un système biomimétique utilisant la technique de « micro-patterning » qui imposent une géométrie d'assemblage pour les MTs dans des limites qui se rapprochent de celles observées dans les cellules. Cette méthode permet de contrôler précisément l'assemblage des MTs à partir de zones dont les formes, la taille et la distribution des unes par rapport aux autres sont définies. Pour valider cette technique, j'ai reconstitué des réseaux qui miment des architectures cellulaires (i.e modules du fuseau mitotique). Dans un deuxième temps j'ai étudié le rôle de MAP65s dans l'auto-organisation de réseaux de faisceaux de MTs, et plus particulièrement l'étape de co-alignement entre MTs dynamiques et dispersés. J'ai montré que MAP65-1 de plante et son orthologue chez la levure, Ase1, diminuent fortement la longueur de persistance de MTs isolés ou organisés en faisceaux. Cet assouplissement leur permet de se déformer et donc de se co-aligner pour former des faisceaux lorsqu'ils se rencontrent à des angles de rencontre élevés. L'augmentation de flexibilité est du à l'interaction du domaine de liaison de MAP65-1/Ase1 avec la lattice des MTs. Ces résultats suggèrent que la diminution de la rigidité des MTs contrôle dans les cellules l'issue des évènements des rencontres entre MTs. De façon plus générale, la modulation des propriétés mécaniques des MTs par des MAPs représente un nouveau mécanisme pour réguler la plasticité des réseaux de MTs dans les cellules eucaryotes. / The microtubule (MT) cytoskeleton is essential for many cell processes, such as the intracellular trafficking, the cell division, and the cell morphogenesis. Depending on the cell type or on its differentiation state, the MT networks will adopt different architectures. These organizations are defined by intracellular geometric constraints and the regulation of the acticity of many MT associated proteins (MAPs). Among these proteins, we get a particular interest in MAP65s family that crosslink MTs to organize them into bundles. The aim of my thesis was to study in vitro the role of MAP65s in the self-organization of MT bundles in particular networks. As a first step, I developed a biomimetic system using the micro-patterning procedure which imposes a MT assembly geometry within limits close to those observed in cells. This method allows to precisely control the MT assembly from micro-patterns with define shape, size and spatial distribution. In order to validate this technic, I reconstituted MT networks which mimic cellular architecture (i.e mitotic spindle modules). In a second time, I studied the role of major MT cross-linkers that are members of the MAP65 family in the formation of MT bundles, particularly the step of MT co-aligment after encountering of dynamic growing MTs. I found that plant MAP65-1 and its yeast ortholog, Ase1, lower the global rigidity of single MTs and MT bundles. This increase in MT flexibility is directly caused by interactions between the MAP65 MT-binding domain and the MT lattice. These data suggest that MT softening by MAP65 controls the issue of MT encounters, so that self-organized ordered MT bundles are formed in living cells. In a more general way, the modulation of MT mechanical propreties by MAPs represent a new mecanism to regulate MT networks plasticity in eukaryote cells.

Biomolecular motor based transport reconstituted in synthetic environment has been recently established as a promising component for the development of nanoscale devices. A minimal system consisting of microtubules propelled over a surface of immobilized kinesin motor proteins has been used to transport and manipulate cargo for molecular sorting, analyte detection, and other novel nanotechnological applications. Despite these achievements, further progress of the field and translation of the reported applications to a real-world setting require overcoming several key challenges, such as, development of effective cargo conjugation strategies and precise control of the transport directionality with the reconstituted biomolecular motor systems. The challenge of cargo conjugation is addressed in this thesis through the development of a robust bioorthogonal strategy to functionalize microtubules. The versatility of the developed method is demonstrated by covalently conjugating various types of cargos to microtubules. Further, the effect of the linker length on cargo attachment to microtubules is investigated by attaching cargo to microtubules via linkers of different lengths. By using kinesin-driven transport of microtubules that are covalently conjugated to antibodies, detection of various clinically relevant analytes is demonstrated as proof-of-principle applications for biosensing. Finally, the challenge of gaining control over transport directionality is addressed through topographical guiding of microtubules in nanostructures, and optimization of assay parameters to achieve successful guiding of microtubules. Spatio-temporal analyte concentration, using transport in these nanostructues, is also explored to make the biomolecular-motor based applications more suitable for use real-world point-of-care setting. Taken together, the experimental work in this thesis contributes to the field of nanotechnological applications of biomolecular motors. The developed microtubule functionalization method and understanding of the effect of cargo attachment via linkers provide useful design principles for efficient cargo loading to microtubules. Moreover, establishment of assay components for successful guiding of microtubules in nanostructures is a vital step forward for practical translation of future nanoscale devices.

Growth anisotropy, in which cells grow predominantly in one direction, is common in plant cells, and an essential event for plant form and function. The direction and degree of growth anisotropy are governed by the mechanical properties of the primary cell wall. When aligned in a parallel manner, cellulose microfibrils accommodate great resistance in the direction of their alignment to expansion driven by isotropic turgor pressure. Using the Arabidopsis thaliana inflorescence stem as a model system, field emission scanning electron microscopy (FESEM) analysis demonstrated that the establishment of parallel arrangement of microfibrils is closely correlated with anisotropic cell expansion. In the novel anisotropy 1 (any1) mutant allele of the primary cellulose synthase CesA1, growth defects were correlated with random cellulose microfibril patterns in some inflorescence stem tissues. Microtubules have been considered to be the most likely candidates for controlling the orientation of cellulose microfibrils. Recent studies have indeed demonstrated a close association of the plasma membrane-localized cellulose-synthase-complexes (CSCs) that produce cellulose and cortical microtubules. Despite this close association, microtubule disruption did not cause cellulose microfibrils to lose parallel alignment in the radial and inner periclinal walls of cells in the inflorescence stem, suggesting that microtubules influence mechanical properties of cellulose microfibrils other than orientation. X-ray diffraction analysis demonstrated that cellulose crystallinity in wild-type plants declines at the growth-promoting temperature of 29°C, whereas crystallinity fails to adapt and remains high in mor1-1, the temperature-sensitive mutant whose microtubule arrays become disorganized at its restrictive temperature (29°C). This finding suggests that organized microtubules are involved in reducing cellulose crystallinity that normally accompanies increased cell expansion. Live-cell imaging of CSCs by tracking a yellow fluorescent protein (YFP)-tagged CesA6 subunit in hypocotyl cells demonstrated that dynamic and well-organized microtubules affect the velocity, the direction of movement, and the density of CSCs, suggesting that there is a close relationship between microtubules and CSCs. Together with the finding that microtubules also control the distribution of COBRA, a GPI-anchored wall protein that is essential for growth anisotropy, I discuss the variety of roles microtubules play in anisotropic growth. / Science, Faculty of / Botany, Department of / Graduate

Le cytosquelette microtubulaire est une des cibles majeures en thérapie anticancéreuse. La caractérisation des isoformes d'une protéine est une problématique complexe en analyse protéomique. Pourtant il est crucial de mettre en évidence les variations de séquence primaire et les modifications post-traductionnelles qui peuvent dans certains cas être corrélées avec un phénomène physiologique et dans d'autres avec un état pathologique. Ainsi, la surexpression de l'isotype βIII de la tubuline, protéine constitutive des microtubules, peut avoir des conséquences importantes sur la régulation des propriétés dynamiques des microtubules et donc sur la réponse des tumeurs aux agents anticancéreux. Chaque isotype de tubuline se distingue principalement par les derniers acides aminés composant leur extrémité C-terminale. L'analyse top-down par MALDI-ISD est une approche de choix pour la caractérisation des isotypes de tubuline dont βIII. De plus, les propriétés spécifiques de fragmentation de cette protéine rendent possible son étude in situ sur coupes de tissue. Le choix de la matrice ainsi qu'une connaissance de la séquence primaire des protéines sont des paramètres importants lors des analyses MALDI-ISD. Une protéine telle qu'EB1, présentant une forte homologie de séquence C-terminale avec l'isotype α1B de la tubuline, est un candidat intéressant pour ce type d'approche. Cette protéine associée aux microtubules est le centre d'un réseau protéique régulant la dynamique des microtubules tel que dans le cas de l'angiogenèse tumorale. L'interaction entre EB1 et les protéines à domaine CAP-Gly se réalise par l'intermédiaire de son motif C-terminal -EEY où la tyrosine semble en être l'élément régulateur. La mise au point d'une approche de protéomique d'affinité pour l'identification et la quantification par spectrométrie de masse des complexe interagissant avec EB1 semble donc être un prérequis pour l'élaboration de nouveaux composés inhibant ce type d'interaction dans des contextes biologiques précis. / Microtubule is one of the major targets in cancer therapy. Protein isoforms characterization is a complex issue in proteomic analysis. Yet is is crucial to highlight primary sequence variations and posttranslational modifications that are associated with physiological processes or pathologies. Thus overexpression of βIII-tubulin isotype, protein that make up microtubules, can have consequences on the regulation of microtubules dynamic properties and therefore tumors response to anticancer agents. Each tubulin isotype is distinguished by the last amino acids of their C-terminus. MALDI-ISD top-down analysis is of interest for tubulin isotypes characterization as βIII. In addition, specific fragmentation properties of this protein make possible its in situ study on tissue sections. Matrix choice and knowledge of proteins primary sequence are important parameters for MALDI-ISD experiments. As protein such as EB1, that presents high sequence homology with α1B-tubulin isotype C-terminus, is an interesting candidate for this kind of approach. This microtubule associated protein is the core of a protein netword that regulates microtubule dynamics such as in the cas of tumor angiogenesis. The interaction between EB1 and proteins with CAP-Gly domain is realized through its C-terminal motif -EEY and tyrosine seems to be the regulatory element. The development of an approach by proteomics affinity for the identification and the quantification by mass spectrometry of complex interacting with EB1 appears to be a prerequisite for the development of new compounds that inhibit this peculiar mode of protein-protein interaction in specific biological contexts.

Adhesion complexes (ACs) are large macromolecular complexes of integrins and associated proteins that connect the actin cytoskeleton to the extracellular matrix. In migrating cells, ACs are highly dynamic -- forming and maturing at the cell front and disassembling at the cell rear. The turnover of ACs enables and localises the necessary traction forces required for cell migration. There is evidence for the spatiotemporal recruitment of specific proteins during AC maturation or disassembly; however, a holistic understanding of the compositional changes to ACs during these states is lacking. To this end, we sought to characterise the dynamic changes that occur at ACs during turnover using a mass spectrometry (MS)-based proteomics approach. A major challenge in studying AC turnover is the desynchronised nature of AC formation, maturation and disassembly within a population of cells. Therefore a nocodazole-washout assay was used to synchronise microtubule-induced AC maturation and disassembly. To study the dynamics of AC turnover by MS, an AC isolation method was optimised for use with the nocodazole-washout assay. Subsequently, the maturation of ACs by the loss of microtubules was studied by MS-based proteomics, and it was found that this resulted in the overall accumulation of adhesion proteins, and also the conversion of fibrillar adhesions to focal adhesions. Studying the dynamic process of AC disassembly requires a sensitive MS quantification method; as such, label-free quantitative methods were compared, and it was found that LC-MS peak ion intensity quantification performed better than spectral counting. Using optimised methodologies for isolation of ACs and MS quantification, the dynamics of AC disassembly was analysed over the course of the nocodazole-washout assay. It was found that in general, microtubules were enriched around ACs, whereas many structural AC proteins decreased over time. In summary, we have optimised methods for the study of ACs by MS-based proteomics, and applied these methods to the study of AC turnover.

Le cancer est l’une des principales causes de mortalité en France, après les maladies cardiovasculaires. Il est responsable de plus de 11 millions de décès dans le monde chaque année. Le cancer résulte d’une prolifération anarchique de cellules qui mène à la formation d’une tumeur. Les cellules tumorales peuvent ensuite migrer vers d’autres tissus pour former des métastases. La chimiothérapie est l’un des traitements les plus utilisés pour traiter le cancer. Elle consiste en l’utilisation d’agents antitumoraux qui provoquent la mort cellulaire en bloquant la mitose. Dans le but d’induire cette apoptose, nous nous sommes intéressés aux poisons du fuseau mitotique, agents cytotoxiques qui ont pour cible les microtubules et qui ont la particularité de se fixer sur leur constituant majeur, la tubuline. La dynamique des microtubules joue un rôle crucial dans la multiplication cellulaire. Bloquer cette dynamique est suffisant pour bloquer la mitose. Par ailleurs, suite à cet arrêt de la polymérisation, un second mécanisme se mettrait en place, notamment au niveau des cellules endothéliales, pour empêcher la néovascularisation, ce qui inhiberait ainsi l’angiogénèse. Notre travail consiste en la conception et la synthèse de nouveaux inhibiteurs de la polymérisation de la tubuline, potentiellement anti-angiogéniques et anti-vasculaires. Il s’agit de tricycles, qui ont la particularité d’interagir spécifiquement avec le site de fixation de la colchicine, au niveau de la tubuline, ce qui inhibe la polymérisation des microtubules et par conséquent la division cellulaire. Des tests d’inhibition enzymatique et de cytotoxicité sur plusieurs lignées cellulaires cancéreuses ont été réalisés et les résultats sont présentés dans ce rapport. / Cancer is one of the leading cause of death in France after cardiovascular diseases. Cancer results from an abnormally excessive cell proliferation which leads to the formation of a tumor. The following processes in invasion of tumoral cells and metastasis in the other tissues. Stopping mitosis by chemotherapy can cause death of cancer cells. In order to induce this apoptosis, we are interested by a class of antitumoral drugs which disrupt mitotic spindle function by focusing on its major component: the microtubules. These agents set exactly on their main structural element: the tubulin. The microtubule was recognised as a subcellular target of major strategic importance with regard to anticancer therapeutics. Our work consisted of the design and the synthesis of new antitumor drugs which influence microtubules dynamics. Theses molecules should inhibit tubulin polymerization by binding to the colchicine site. Moreover, we expect theses compounds selectively target tumor vasculature and thus can also be considered vascular disrupting agents. Enzymatic inhibition and cytotoxic assays were performed on different cell lines and are presented in this report.