Les microtubules sont des filaments protéiques dynamiques, essentiels au bon fonctionnement de la plupart des cellules eucaryotes. L’organisation de ce réseau de fibres constitue un enjeu majeur pour la cellule, puisque c’est de cette organisation que découle une grande partie de ses fonctions. Dans les cellules animales, la protéine EB1 (End Binding-1) est impliquée dans la polarisation du réseau de microtubules. Arabidopsis thaliana possède trois gènes orthologues bien conservés, mais dont les fonctions sont encore mal connues, alors même que l’orientation et l’organisation du réseau de microtubule sont critiques pour le développement des plantes. En particulier, dans les cellules en élongation, le réseau cortical de microtubules est perpendiculaire à l’axe de croissance et permet, en participant à la synthèse de la paroi, de restreindre la croissance en épaisseur au profit d’un allongement. Les microtubules corticaux ne sont pas isolés les uns des autres, ils s’associent latéralement pour former des faisceaux, ajoutant ainsi un niveau de complexité, et donc de régulation, à l’architecture du réseau microtubulaire chez Arabidopsis thaliana. La formation et la maintenance du réseau de faisceaux parallèles de microtubules constituent l’objet principal de ce travail de thèse. Chez Arabidopsis thaliana, EB1 est impliquée, à l’échelle macroscopique, dans la croissance directionnelle des racines. Toutefois, les fonctions subcellulaires de la protéine étaient peu connues au début de nos investigations. Tout d’abord, notre étude a permis de montrer que le réseau cortical de microtubules est désorganisé chez des plantes mutantes dépourvues de protéine EB1 cytoplasmique. De plus, la combinaison de la microscopie super-résolue STED et d’une procédure d’analyse d’image que nous avons élaborée au laboratoire, a mis en évidence une diminution significative du nombre de microtubules par faisceau en absence de EB1. Nous avons également observé une hypersensibilité des racines mutantes à la dureté du milieu, confirmant des données publiées précédemment. Pris dans leur ensemble, ces résultats suggèrent : (1) l’importance de l’organisation du réseau cortical de microtubules dans la réponse de la racine au toucher ; (2) une probable interdépendance entre organisation du réseau et formation des faisceaux. Ensuite, afin de confirmer le probable lien fonctionnel entre la formation des faisceaux de microtubules et organisation globale du réseau microtubulaire cortical, nous avons étudié des plantes mutantes pour MAP65-1, une protéine déjà décrite pour sa capacité à former des faisceaux in vitro. Nos premiers résultats, tendent à confirmer cette fonction de MAP65-1 in vivo et révèle, pour la première fois, une implication significative de cette protéine dans l’arrangement parallèle des microtubules corticaux. Si ce résultat ne met pas en évidence la relation de cause à effet qui relie ces deux phénomènes, il confirme toutefois l’existence d’un lien entre les deux niveaux de régulation. Enfin, dans le but de mieux comprendre les mécanismes permettant aux protéines EB1 et MAP65-1 de former des faisceaux de microtubules, nous avons entamé une analyse de leurs propriétés intrinsèques in vitro, en système purifié. Les premiers résultats, très préliminaires, indique un effet stimulateur de EB1 sur la capacité de MAP65-1 à former des faisceaux de microtubules. Cette thèse a contribué à la compréhension des mécanismes qui régissent l’organisation du réseau de microtubules corticaux chez Arabidopsis thaliana, incluant la formation des faisceaux de microtubules et le rôle joué par EB1 et MAP65-1 dans ce contexte. Elle confirme également l’implication du réseau de microtubules dans le contrôle de la croissance racinaire et suggère fortement sa participation à la réponse aux contraintes mécaniques. / Microtubules are essential dynamic filaments of most eukaryotic cells. Microtubule network organization is tightly controlled within cells since most of microtubule functions come from their spatial arrangement. In animal cells, EB1 (End Binding-1 protein) is well known as a major regulator of microtubule network polarization. Though well conserved throughout evolution, Arabidopsis thaliana possesses three EB1 orthologous genes with unclear functions, while microtubule network orientation and organization are critical for plant development. During plant cell expansion, cortical microtubules are organized as parallel fibers that are perpendicular to the elongation axis. This particular organization is thought to promote cell elongation rather than thickening by controlling cell wall synthesis. Cortical microtubule are not isolated from each other, they are laterally associated within bundles, bringing an additional level of complexity, and therefore of regulation, to the microtubule network in plants. Microtubule bundles formation and maintenance are the main interest of this PhD-thesis work. In plants, EB1 proteins had already been involved in directional root growth, but their subcellular functions remained unclear. Our study revealed first that the cortical microtubule network is disorganized in plants lacking cytoplasmic-EB1 protein. Moreover, using super-resolution microscopy combined with an original image processing, we showed that the average number of microtubules per bundle is significantly reduced in the absence of EB1. In addition, EB1-defective roots display a hypersensitivity to medium hardness as mentioned elsewhere before. Altogether, our data suggest: (1) an involvement of the microtubule network in root response to touch; (2) a possible relationship between microtubule-network organization and bundle formation. Then, in order to confirm the functional link between bundle formation and network organization, we tackle the study of MAP65-1 mutant plants. MAP65-1 is a protein well described for its ability to make microtubule bundles in vitro. Our investigations confirmed this function for MAP65-1 in vivo and reveal its involvement in cortical microtubule network organization. Although this result does not reveal any causal connection between both phenomena, it highlights the link between the two levels of complexity that are bundle formation and spatial arrangement of microtubules. Finally, to get insight into the molecular mechanisms allowing EB1 and MAP65-1 to make microtubule bundles, we developed in vitro experiments using purified components. Preliminary results indicate that EB1 stimulates MAP65-1 ability to make bundles, but this remains to be further investigated. Hence, this thesis work contributed to decipher the mechanisms governing microtubule network organization in Arabidopsis thaliana. In particular, it revealed the involvement of EB1 proteins and MAP65-1 in this task. This work further confirmed the role of microtubules in root growth and strongly suggested their involvement in the response to mechanical sensing.

No description available.

Dendrites of cockroach tibial spine mechanoreceptors contain hundreds of free microtubules, which may have some relation to the generation of electrical activity. Deflection of a spine produces a train of action potentials. Continuous perfusion over a period of 4 hours results in no response decrement. Perfusion with 10mM colchicine reversibly inhibits the response within 5-7 minutes. Irreversible inhibition is produced by perfusion with 1mM vinblastine sulfate in perfusion solution containing 1% dimethyl sulfoxide. Deuterium oxide does not inhibit at concentrations less than 50%, nor does it counteract inhibition by 10mM colchicine. Colchicine may be affecting (1) intracellular microtubules, (2) membraneous tubulin, (3) other membrane components, or (4) axoplasmic transport of essential materials to the sensory dendrites.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Microtubules in the fungal pathogen Ustilago maydis have important roles, which include polar budding, morphogenesis and nuclear migration. They also serve as tracks for molecular motors, responsible for intracellular transport of organelles and membrane trafficking. Moreover, microtubules are indispensable during both interphase and cell division, and they play a crucial role in long-distance microtubule-based transport, which occurs in neurons or fungal hypha. Therefore, in order to carry out their functions correctly they need to be well organised and stabilised, which is achieved mainly by various microtubule-associated proteins. In this thesis, different aspects of microtubule (MT) cytoskeleton organisation in U. maydis were investigated, using bioinformatics and experimental approaches. In the first part of the thesis I studied the microtubule-associated protein (MAP) repertoire in U. maydis, which has never been done before in a comprehensive way. For this purpose, searches across five eukaryotic model organisms were conducted to identify all of their known MAPs, to query the U. maydis database. In addition, all of the proteins were checked for their domain architecture, to help decide if an orthologue had been found. As a result, 66 potential MAP orthologues were identified. The second part of this thesis focused on identifying novel factors involved in the organisation of the microtubule cytoskeleton using a specially designed genetic screen. This work involved five microtubule-organisation defect (MOD) mutants, generated by UV-mutagenesis, which were characterised by inability to produce long hyphae as well as by short, fragmented microtubules. To find which genes were responsible for this phenotype, the genomes of all mutants were sequenced and compared with a wild-type genome, and mutations in many genes were found. The analysis revealed potential candidate genes responsible for the specific phenotype of the mutants. However, most probably, UV-generated point mutations in more than one gene played a part in the defective microtubule array. In the final part of this thesis, the function of two beta-tubulin isotypes in U. maydis was analysed. Using conditional mutants, I demonstrated that there are subtle functional differences between the two beta tubulins.

Pinus radiata is by far the dominant species grown in New Zealand plantations as a renewable source of wood. Several wood quality issues have been identified in the material produced, including the high incidence of compression wood, which is undesirable for end users. At present our understanding of the complex array of developmental processes involved in wood formation (which has a direct bearing on wood quality) is limited. Hence, the forest industry is interested in attaining a better understanding of the processes involved. Towards this goal, and for reasons of biological curiosity, the experiments described in this thesis were carried out to investigate several aspects of xylem cell development. In an in arbor study, changes in the orientation of cortical microtubules and cellulose microfibrils were observed in developing tracheids. Results obtained provide evidence that cortical microtubules act to guide cellulose synthase complexes during secondary wall formation in tracheids. The mechanisms involved in controlling cell wall deposition in wood cells are poorly understood, and are difficult to study, especially in arbor. A major part of this thesis involved the development of an in vitro method for culturing radiata pine wood in which hormone levels, nutrients, sugars and other factors, could be controlled without confounding influences from other parts of the tree. The method developed was used in subsequent parts of this thesis to study compression wood development, and the influence of the hormone gibberellin on cellulose microfibril organisation in the cell wall. Results from the in vitro compression wood experiments suggested that: 1. when a tree is growing at a lean, the developing cell wall was able to perceive compressive forces generated by the weight of the rest of the tree, rather than perceive the lean per se. 2. ethylene, rather than auxin, was involved in the induction of compression wood. Culture of stem explants with gibberellin resulted in wider cells, with steeper cortical microtubules, and correspondingly steeper cellulose microfibrils in the S2 layer of developing wood cells. This observation provides further evidence that the orientation of microtubules guides the orientation of cellulose microfibrils. Overall, the work described in this thesis furthers our knowledge in the field of xylem cell development. The stem culture protocol developed will undoubtedly provide a valuable tool for future studies to be carried out.

<p>Microtubules often adopt non-centrosomal arrays in differentiated tissues, where they are important for providing structure to the cell and maintaining polarity. Although the formation and organization of centrosomal arrays has been well-characterized, little is known about how microtubules form non-centrosomal arrays.</p><p>In the mouse epidermis, centrosomes in differentiated cells lose their microtubule-anchoring ability through the loss of proteins from the centrosome. Instead, microtubules are organized around the cell cortex. The cell-cell adhesion protein desmoplakin is required for this organization. Our model is that desmoplakin recruits microtubule-anchoring proteins like ninein to the desmosome, where they subsequently recruit and organize microtubules.</p><p>To test this model, we confirmed that the microtubule-binding proteins Lis1, Ndel1, and CLIP170 are recruited by desmoplakin to the cell cortex. Furthermore, by creating an epidermis-specific conditional Lis1 knockout mouse, I found that Lis1 is required for cortical microtubule organization. Surprisingly, however, Lis1 is also required for desmosome stability. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.</p><p>Although Lis1 is required for microtubule organization, it is not sufficient. I created a culture-based system to determine what other factors may be required for cortical organization for microtubules. My work reveals that stabilization of the microtubules is sufficient to induce their cortical organization. Functionally, cortical microtubules are important for increasing the mechanical integrity of cell sheets by engaging adherens junctions. In turn, tight junction activity is increased. Therefore, I propose that cortical microtubules in the epidermis are important in forming a robust barrier by cooperatively strengthening each cell-cell junction.</p><p>To determine whether desmosomes play similar roles in simple epithelia as stratified epithelia, I examined intestinal epithelial-specific conditional desmoplakin conditional knockout mice. Unexpectedly, I found that desmoplakin is not required for cell-cell adhesion and tissue integrity in the small intestine. Furthermore, it does not organize intermediate filaments. Desmoplakin is required, however, for proper microvillus architecture. </p><p>Overall, my studies highlight novel tissue-specific roles for desmosomes, in particular desmoplakin, in organizing and integrating different cytoskeletal networks. How desmoplakin's function is regulated in each tissue will be a new interesting area of research.</p> / Dissertation

Contexte: Le centrosome est un petit organite bien connu pour son rôle dans l'établissement du fuseau bipolaire pendant la division cellulaire. Les déficiences de la fonction du centrosome donnent souvent lieu à des maladies humaines, y compris le cancer et la formation de kystes rénaux. Nous sommes intéressés à étudier la fonction d'une nouvelle protéine centrosomale nommée CEP78, identifiée dans un criblage protéomique pour de nouveaux composants centrosomaux. Méthodes et résultats : Le traitement des cellules avec le nocodazole, un agent qui dépolymérise spécifiquement les microtubules cytoplasmiques mais pas les microtubules stabilisés du centrosome, a montré que CEP78 est un composant centrosomal stable. La colocalisation de cette protéine avec d'autres marqueurs centrosomaux tels que CEP164, SAS6, Centrine, tubuline polyglutamylée et POC5, à différentes phases du cycle cellulaire a indiqué que CEP78 est précisément à l'extrémité distale des centrioles, mères et filles. Il existe deux pointts CEP78 au cours de l’interphase et les cellules passent par la mitose, procentrioles maturent, et le nombre de points de CEP78 augmente à 4 par cellule et, à la fin de la télophase chaque cellule fille possède 2 points CEP78. La caractérisation des domaines fonctionnels de CEP78 a montré que des répétitions riches en leucine sont nécessaires pour la localisation centrosomale de la protéine. En outre, nous avons constaté que la surexpression de CEP78 ne change pas le nombre de mères/procentrioles mais diminue le nombre et l'intensité des points de CEP170 (protéine d'appendice sous-distal) sans diminution du niveau d'expression de cette protéine. D'autres études ont montré qu'il n'y a pas d'interaction entre ces deux protéines. Enfin, la surexpression de CEP78 protège des microtubules contre la dépolymérisation en présence de nocodazole, ce qui suggère qu'il possède la capacité de lier les microtubules. Conclusion : Nos résultats suggèrent que CEP78 est destiné à l'extrémité distale des centrioles matures par ses répétitions riche en lecuine, où il pourrait être impliqué dans la maturation ou la régulation de l'assemblage ou de la rénovation de l'appendice sous-distal centriolaire, une structure connue dans la nucléation des microtubules et d'ancrage. Comprendre la fonction de Cep78 contribuera à éclaircir le rôle du centrosome dans le cycle cellulaire. / Background: The centrosome is a tiny organelle well-known for its role in establishing the bipolar spindle during cell division. Defects in centrosome function often give rise to human diseases including cancer and kidney cyst formation. We are interested in studying the function of one novel centrosomal protein named CEP78, identified in a proteomic screen for novel centrosomal components. Methods and results: Treatment of cells with nocodazole, a microtubule-depolymerizing agent that specifically depolymerizes cytoplasmic microtubules but not the stabilized centrosome microtubules, showed that CEP78 is a stable centrosomal component. Colocalization of this protein with other centrosomal markers such as CEP164, SAS6, Centrin, Polyglutamylated tubulin and POC5 at different phases of the cell cycle indicated that CEP78 specifically localizes to the distal end of the mother and daughter centrioles. There are 2 CEP78 dots during the interphase and as the cells go through mitosis, procentrioles mature, and the number of CEP78 dots increases to 4 dots per cell and by the end of telophase each daughter cell has 2 CEP78 dots. Characterization of CEP78 functional domains showed that Leucine-rich repeats are necessary for centrosomal localization of the protein. In addition, we found that overexpression of CEP78 did not change the number of centrioles and centrosomes but decreased the number and intensity of CEP170 dots (sub-distal appendage protein) without a decrease in the expression level of this protein. Further studies showed that there is no interaction between these 2 proteins. Finally, overexpression of CEP78 protects microtubules from depolymerization in the presence of nocodazole, suggesting its ability to bind microtubules. Conclusion: Our findings suggest that CEP78 is targeted to the distal end of mature centrioles via its lecuine-rich repeats, where it could be involved in centriolar maturation or regulation of sub-distal appendage assembly and/or remodeling, a structure known to nucleate and anchor microtubules. Understanding the function of CEP78 will shed light on the role of the centrosome in cell cycle.

Microtubular cytoskeleton is involved in many processes in plant cells, including cell division, growth and development. Other proteins enable its functions by modulation of its dynamics and organization and by mediation of functional and structural interaction with other cell structures. Identification of the mediating proteins and the functions of these interactions under specific conditions were the main aims of the thesis. Membrane proteins interacting with microtubules were identified using biochemical methods. Surprisingly, the identified proteins co-sedimenting with microtubules were not members of the "classical" microtubule associated proteins (MAPs). There were enzymes, chaperones and plant specific proteins among them. For further studies, the identified microtubule-associated heat-shock protein 90 (Hsp90_MT) was chosen. Recombinant Hsp90_MT binds directly to microtubules and tubulin dimers in vitro. The ATP-binding pocket is not responsible for this association. In BY-2, Hsp90_MT co-localizes with phragmoplast and cortical microtubules and is involved in microtubule recovery after their depolymerization during cold treatment. In plants, Hsp90 is involved in cell cycle progression, its inhibition causes cell-cycle arrest in G1 phase. Based on literature search for animal proteins...

Biological systems are intricate self-assembled systems built from dynamic nanoscale components. These nanoscale components are responsible for many tasks, from subcellular (e.g. DNA replication, cytoplasmic streaming, intracellular transport) to organismal (e.g. intercellular signalling, blood circulation). At each level, biological materials demonstrate complex and dynamic behaviors which are still robust to many perturbations, requiring a balance of dynamism and stability. Being able to emulate biology by dynamically assembling complex systems and structures from nanoscale building blocks would greatly expand the types of materials and structures available, possibly leading to better smart, adaptive, self-healing materials in engineering. The overarching goal of this dissertation is to further the understanding of assembly in dynamic nanoscale systems. To this end, in vitro assays of kinesin motor proteins and microtubule cytoskeletal filaments are employed, providing a well-tested, minimalist, and convenient model system. In these assays, the kinesin motors are attached to the surface of the flow cell and the microtubule filaments are propelled over them. As the majority of past studies in active self-assembly of microtubules have been performed with biotin-labeled microtubules with streptavidin as a cross-linker (a "sticky" gliding assay), the first three parts of this dissertation focus on that system. In the first part, the adsorption kinetics of the streptavidin cross-linker onto the microtubule, which determines the interaction strength between microubule building blocks, is studied. The adsorption curve suggests that this is a negatively cooperative process, and here, the cause of the apparent negative cooperativity in the adsorption process is elucidated as a combination of steric and electrostatic interactions. In the second part, the difference between kinesin-propelled assembly and diffusion-driven assembly is investigated. While the kinesin-propelled microtubule assay has been used for over a decade, a control experiment comparing the active motor-driven system to a passive diffusion-driven system had never been performed. The control experiments showed conclusively that the passive system resulted in smaller and more disordered structures. Furthermore, these results fit well with existing models. The third part investigates the origins of microtubule spools observed in kinesin-propelled microtubule gliding assays, where the microtubules are allowed to cross-link via streptavidin and biotin. These microtubule spools have long been considered an example of a non-equilibrium structure which arises in motor-driven assembly. These spools exist in a dynamic state, having been observed to unwind in previous studies, and store large amounts of bending energy. Determining the origins of these spools is a first step towards understanding how to induce dynamically stable states. Finally, in the last part, a new dynamic system is engineered in which the microtubule assembles its own kinesin track as it moves along the surface while kinesin tracks which are not being used spontaneously disassemble. Thus, this system is stable enough to promote the motion of microtubules over the surface, but dynamic enough to allow for components to be recycled and assembled as needed. While such systems have been realized with mesoscopic to macroscopic components, such a system had not been realized in the nanoscale. As such, the realization of this system is the first step towards designing biomimetic active materials. Throughout this dissertation, the importance of short-range interactions on assembly kinetics is highlighted. The findings presented not only further the understanding and theory behind self-assembly in active nanoscale systems, but also further push the boundaries of experimentally realized systems.