New aspects of shape control and fibre deployment are reported for three situations: certain insect ovarian follicles, certain insect wings, and an unusual nucleus, the ciliate micronucleus. The shaping of insect follicles includes the spatio-temporal integration of intracellular and extracellular fibre arrays in the cockroach follicle. The situation is more complex than previously supposed. Similar events occur in Rhodnius prolixus follicles. It is argued, largely on the basis of my survey, that all insect follicles are involved in the control of egg shape. Evidence is also presented for follicular re-organisation and involvement as a contractile tissue during egg discharge. This possibility has never been considered before. It involves a previously undetected post-vitellogenic phase of cytoskeletal co-ordination. Epidermal cells exhibit a striking sequence of very marked changes in shape during wing morphogenesis in the dipteran insect Calliphora erythrocephala. This includes two epithelial cell contraction-elongation cycles that are spatio-temporally co-ordinated and apparently help to define the shape of a growing wing. It has been shown for Paramecium that an unusual type of spindle microtubule differentiation is involved in micronuclear mitosis. This microtubule differentiation occurs with remarkable spatial and temporal precision at specific locations within the spindle at specific stages in micronuclear elongation.

The morphogenetic movements in epithelial sheets seen during organogenesis are driven in part by changes in cell shape, which in turn depend on changes in the cytoskeleton. In this thesis I used the formation of the salivary glands in the Drosophila embryo, which are formed by coordinated apical constriction and invagination of a polarised epithelial placode, as a model to understand these changes. The role of microtubules (MTs) during morphogenetic processes such as this are not well understood. In order to ascertain their role in successful tubulogenesis I looked at both stable and newly polymerized MTs within the salivary gland placode. Apical constriction is accompanied by the loss of a centrosomal apically localised MT network and the appearance of a longitudinal acentrosomal MT network. In parallel, MTs become increasingly more stable, starting from the apical surface and spreading more basally, leading to stable MT bundles that appear nucleated and anchored near the apical surface independent of centrosomes. Disrupting the MTs specifically in the salivary gland placode using the MT-severing protein Spastin results in defects in apical constriction, and at later stages tube lumen defects. Wild type cells generate an apical medial actomyosin meshwork that likely drives apical constriction. MT ends present at the apical surface colocalise with apical medial actomyosin, and cells lacking MTs fail to maintain apical medial actomyosin. This indicates crosstalk between the MT and actomyosin cytoskeleton during apical constriction and tissue bending. The cytolinker protein Short Stop (Shot) has previously been shown to be able to link the actin and MT cytoskeleton. Shot rearranges concomitant with the MT cytoskeleton and colocalises with both with the tips of MT bundles and apical medial actin. Further to Shot, the formin Diaphanous regulates both actin and microtubules in morphogenesis, and was also found to localize to MT tips at the apical surface. Overexpression of constitutively active Diaphanous results in precocious invagination of the salivary gland. Overall this suggests a role for acentrosomal MT bundles in maintaining an apical actomyosin meshwork for successful apical constriction and subsequent tubulogenesis. Cytoskeletal crosstalk may be mediated by the cytolinker Shot and the formin Diaphanous.

The molecular dynamic of tau protein, its interaction with microtubules and the changes in both that appear in pathological conditions are a focus of research to get an insight in neurodegeneration. It is known that tau binds to microtubules by four homologous repeats in its carboxyterminal half. We want to examine tau and especially its repeat regions and their role for microtubule-interaction in living neurons using live cell imaging. We created multiple tau fragments with different numbers of repeats and expressed them in neuronal differentiated PC12 cells as a model for neurons. We performed fluorescence decay after photoactivation experiments by measuring the change of fluorescence intensity over time in the activated region in the middle of cell processes. From this experiments we were able to determine association and dissociation rates of the respective constructs by fitting and modeling approaches. Fluorescence decay increased with decreasing number of repeats. We found that a minimum number of three repeats required for microtubule interaction. This could also be observed in the tip of the processes. Destabilization of microtubules by colchicine increases the mobility of full length tau and microtubule-interacting fragments, while stabilization with epothilone D has no effect. Pseudophosphorylation of tau does not significantly affect the fluorescence decay, but leads to an increase of the dissociation and association rate. Truncation of the carboyxterminus after amino acid 421 - which simulates caspase 3 cleavage of tau - or amino acid 401 leads to a decrease of fluorescence decay indicating increased binding of these fragments to microtubules and a higher dissociation rate of these fragments. Furthermore we could show that overexpression of full length tau in PC12 cells increases the fraction of polymerized tubulin. The stronger binding caspase cleavage fragment shows a similar microtubule stabilization.

Les microtubules sont des éléments du cytosquelette, composées d'hétérodimères de tubuline de type α et β. Ils jouent un rôle important dans plusieurs processus cellulaires, dont le transport cytoplasmique, la mobilité et la division cellulaire. Cependant, les mécanismes par lesquels les microtubules sont adaptés à ces rôles très différents restent largement méconnus.Les modifications post-traductionnelles de la tubuline pourraient contribuer à la diversité de fonction des microtubules. Parmi celles-ci, la polyglutamylation pourrait jouer un rôle important en changeant d'une manière importante les propriétés des microtubules, et les adaptant ainsi à leurs différents rôles. La polyglutamylation correspond à l'addition de longues chaînes latérales d'acides glutamiques aux extrémités C-terminales des tubulines α et β. Ces régions sont des sites connues d'interactions de la tubuline avec ses protéines associées (MAP) et les moteurs moléculaires. Au cours de mes travaux, j'ai étudié le rôle de polyglutamylation de la tubuline dans le développement des spermatozoïdes. En utilisant la souris et la drosophile comme organismes modèles, j'ai montré que le changement de niveau de polyglutamylation de la tubuline dans les spermatozoïdes, soit par une régulation positive soit par régulation négative, provoque des anomalies structurales des spermatozoïdes et entraîne une stérilité des mâles. J'ai également étudié la rôle de la katanine, un enzyme coupant les microtubules, effecteur potentiel de la polyglutamylation. J'ai montré qu'en absence de la katanine, la production des cellules germinales est gravement compromise chez les mâles, provoquant également une stérilité. Pris ensemble, ces résultats démontrent que la régulation de polyglutamylation de la tubuline est indispensable pour le développement correct des spermatozoïdes et que son effet pourrait être médié par la katanine, enzyme dont l'activité pourrait dépendre de la polyglutamylation de la tubuline.Durant mes travaux, j'ai également développé une nouvelle technique de production des microtubules différentiellement glutamylés. En utilisant comme matière primaire de la tubuline de cerveau porcin, hautement polyglutamylée, j'ai réalisé une déglutamylation produisant ainsi une tubuline déglutamylée. En utilisant les deux types de microtubules (avec ou sans polyglutamylation), il est possible de tester si les interactions entre les microtubules et les MAP dépendent de la polyglutamylation de la tubuline. / Microtubules are essential cytoskeletal elements composed of α- and β-tubulin heterodimers. They are involved in a number of cellular processes, including intracellular transport, cell motility and cell division. However, how microtubules can adapt to all these different functions remains largely unknown. One of the mechanism, which could contribute to microtubule diversity are posttranslational modifications of tubulin. Among tubulin modifications polyglutamylation has a high potential for changing microtubule properties and thus adapting them to different roles. It consists of addition of long glutamate side chains to multiple glutamate residues located in the C-terminal tail of both α- and β-tubulin, which are known as interaction sites for many microtubule associated proteins (MAPs) and molecular motors. In my studies I focused on the role of polyglutamylation in sperm development. Using mice and Drosophila as model systems, I showed that changing the levels of this modification, either by up- or downregulation, results in the assembly of structurally abnormal sperm and causes male sterility. In addition, I also addressed the role of one of the potential effectors of polyglutamylation, a microtubule-severing enzyme called katanin. I demonstrated that in the absence of katanin the production of male germ cells is severely compromised leading to male sterility. Taken together my data suggest that proper balance of tubulin polyglutamylation is essential for sperm development and that its effects may be mediated by katanin whose activity has been proposed to be dependent on tubulin polyglutamylation. Moreover, during my PhD project I developed a method for production of differentially glutamylated microtubules. Using porcine brain tubulin, which is known to be highly glutamylated, as a starting material I perform deglutamylation to obtain the non-glutamylated version of it. Obtaining these two types of tubulin allows now to directly testing whether the interactions between microtubules and the MAPs of interests are dependent on tubulin polyglutamyaltion.

Thesis (Ph.D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on August 13, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medical Sciences - Biomedical Engineering, University of Alberta." Includes bibliographical references.

Les microtubules (MT) sont des polymères dynamiques ancrés par leurs extrémités moins aux centres de nucléation alors que leurs extrémités plus, explorent le cytoplasme, jusqu’à être stabilisées. Cette capture des extrémités permet l’organisation du réseau des MT. Les +TIP sont un groupe de protéines qui s’associent aux bouts plus des MT. EB1 est une protéine centrale dans le réseau des +TIP qui régule la dynamique des MT et leur interaction avec les structures d’ancrage des extrémités plus. Par protéomique ciblée, nous avons caractérisé l’interactome d’EB1, et mis en évidence un groupe de protéines, précédemment associées aux centres de nucléation incluant AKAP9, une protéine échafaudage pour les protéines kinases A (PKA), la protéine de la matrice péricentriolaire CDK5RAP2, et une isoforme courte de la myomégaline que nous avons appelé SMYLE (Short MYomegalin Like EB1 binding protein). La cartographie moléculaire a permis de montrer que ces protéines formaient un complexe organisé de manière hiérarchique. Nous avons observé que l’association transitoire deLa protéine SMYLE (Short MYomegalin Like EB1 binding protein )dans l'organisation d'un complexe centrosomal, la régulation de la nucléation et la stabilisation des microtubules : conséquences sur la migration et la division des cellules cancéreuses avec les MT néo-nucléés au centrosome favorisait la nucléation et l’acétylation des MT. De manière notable, la déplétion de SMYLE aboutissait à un défaut de nucléation, mais aussi de la capture corticale des MT. Ces défauts dans l’organisation des MT étaient associés à une baisse notable de la migration des cellules de carcinome mammaire et à des anomalies mitotiques. Nos résultats nous permettent de proposer que SMYLE fait partie d’un complexe centrosomale, qui favorise l’assemblage ou la stabilité des microtubules néo-nucléés, contribuant ainsi à des processus majeurs pour le développement tumoral. / Microtubules (MT) are dynamic polymers anchored by their minus ends at the MT organizing centers while their highly dynamic plus end explores the cytoplasm until it get stabilized. This plus end capture allows the organization of the MT network. +TIPs are a group of proteins that share the commonality to associate either directly or indirectly to MT plus ends. EB1 is a central protein of the +TIP network that regulates MT dynamics and their interactions with plus end anchoring structures. Using targeted proteomics, we have characterized the EB1 interactome and revealed a set of protein previously shown to associate with the nucleating centers that included AKAP9 an anchoring protein for protein kinase A (PKA), the pericentriolar matrix protein CDK5RAP2 and a short Myomegalin isoform that we named SMYLE (Short MYomegalin Like EB1 binding protein). Molecular mapping revealed that the proteins formed a hierarchically organized complex. We have observed that the transient association of SMYLE to the newly nucleated MTs at the centrosome favored the nucleation and acetylation. Interestingly, SMYLE depletion led to MT nucleation defects, but also a disruption of cortical MT capture. These defects in the MT network were associated with a steep fall in the migratory potential of breast cancer cells and mitotic abnormalities. Our results allow proposing that SMYLE belongs to centrosomal supramolecular complex that favors the assembly and stability of newly nucleated MTs, thus contributing to major processes in tumor development.

Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.

La spermatogenèse est le processus par lequel les cellules germinales sont transformées en spermatozoïdes par le déroulement de 3 phases: la phase mitotique et méiotiques et la spermiogénèse. Pendant la spermiogénèse d'importantes structures sont formées afin de générer un spermatozoïde fonctionnel : l’acrosome, la manchette et le flagelle. La manchette est une structure transitoire situé caudalement à l’acrosome, composée par un manteau de microtubules longeant le noyau du spermatide. La manchette est connue pour participer au remodelage du noyau afin de lui conférer une forme falciforme ainsi que pour son rôle dans le développement de l’acrosome et du flagelle. En effet, pendant la spermiogénèse toutes les molécules nécessaires pour la formation du flagelle et de l’acrosome doivent être transportées sur leur site d'assemblage. Les microtubules forment la manchette permettent le mouvement de protéines entre la région pré-acrosomique et la zone d’assemblage du flagelle. Cependant ce transport doit être finement régulé dans l’espace et dans le temps car la localisation aberrante et/ou manquante de certaines protéines peut causer des malformations de l’ acrosome, de la manchette et du flagelle. Un mécanisme qui peut expliquer la façon dont ce processus de transport peut être régulé est la génération de modification post-traductionnelles de la tubuline forment les microtubules car ces modifications peuvent réguler les interactions avec les moteurs moléculaires et les protéines associées aux microtubules. La polyglutamylation correspond à un attachement covalent de chaines de glutamates latérales sur la queue terminale de la tubuline. Cette modification est contrôlée par la coordination des enzymes glutamylase (TTLLs) et déglutamylase (CCPs). De récents études ont souligné l'importance potentielle de certaines de ces enzymes dans la formation et la maintenance du flagelle. Mon projet est centré sur l’étude des fonctions exercées par CCP5 pendant la spermatogenèse chez la souris. CCP5 est le seule enzyme qui a la capacité de couper le glutamate de branchement des chaines latéral et qui peut donc réguler l’équilibre entre présence ou l’absence de glutamate de branchement. L’analyse de la souris CCP5-knockout a permis de souligner le rôle essentiel mené par CCP5 pendant la spermiogénèse. J'ai constaté que les souris CCP5-KO produisent 100 fois moins de sperme, défectueux et immobile, comparé aux contrôles. De plus, des nombreuses cellules haploïdes immatures sont prématurément libérées de l’épithélium germinatif. Une analyse approfondie à révélée que la réduite production de sperme est due à plusieurs défaut ultrastructurelles qui surgissent pendant la spermiogenèse. J’ai observé que l’acrosome n’était pas bien développée et que cela se détachait du noyau chez les spermatides matures condensés. De plus l’organisation des microtubules formant la manchette était aussi affectée par une émanation ectopique, ainsi que par une localisation défectueuse dans le noyau. Ces défauts corrèlent avec la formation des spermatides allongée que n’ont pas la typique forme falciforme. De plus, j’ai constaté la présence de centrioles surnuméraires chez les spermatides allongées CCP5-KO. Ce défaut corrèle avec l’observation de microtubules « doublets » et « singlets » dispersés dans le cytoplasme de la cellule. De plus, les structures accessoires du flagelle se positionnaient, de façon désorganisé, à côté de ces microtubules. On a pu constater que ces microtubules sont très probablement issus de plusieurs axonemes qui s'ouvrent dans leur région. Le processus entier de spermiogenèse semble être défectueux dans la souris CCP5-KO et cela est accompagné par d'importants changements de niveaux de glutamylation chez les spermatides rondes et allongés. Par conséquence la régulation des niveaux de glutamylation faites par CCP5 lors de la spermiogénèse semble être fondamental pour garantir un développement normal des spermatides en spermatozoïdes. / Spermatogenesis is the process by which germ cells are transformed into spermatozoa by three sequential phases: the mitotic- and meiotic- phase followed by spermiogenesis. To allow the final maturation of haploid germ cells into spermatozoa specific structures have to be developed during the spermiogenesis: the acrosome, the manchette and the flagellum. The manchette is a MTs-based structure, located caudally to the acrosome, organizing in a skirt-like fashion. Manchette is known to participate in the shaping of the nucleus conferring it the typical hook-like shape and several studies have underlined its importance in acrosome and flagellum formation. During spermiogenesis all molecules and organelles necessary for both acrosome and flagellum formation have to be transported to their destination sites and manchettal MTs allow the movement of organelles and other proteins between the pro-acrosome region and the spermatid tail. However this MTs-based traffic has to be regulated both in space and time as it has been shown that ectopic or mislocalization of certain proteins can lead to failures in acrosome, manchette and flagellum development. The generation of posttranslationally modified MTs might explain a possible mechanism of traffic regulation since it has been demonstrated that posttranslational modifications (PTMs) can regulate the interaction between MTs and molecular motors and microtubules binding proteins. Polyglutamylation, consist in the addition of glutamate side chains of variable length on α- and β- tubulin carboxy-terminal tails. Glutamylation levels are determined by the combined action of glutamylase (TTLLs) and deglutamylase (CCPs) enzymes. Several reports have recently highlighted the importance of some of these enzymes in flagellum assembly and/or maintenance. During my PhD I investigated about the functional role of CCP5 during mouse spermatogenesis. CCP5 is the only enzyme able to remove the glutamate branching point of the added side chain. Thus, its activity might regulate the equilibrium between presence/absence of glutamate branching points, in turn interfering with polyglutamylation levels. The study of the CCP5-KO mouse reveals that CCP5 has an essential role during mouse spermiogenesis. CCP5-KO male produces 100-fold less sperm cells than controls and released sperm cells are highly defective and immotile. Moreover, haploid immature germ cells are also found in CCP5-KO semen. A deep-analysis reveals that the reduced sperm output is due to several ultrastructural defects emerging during the spermatids differentiation process. The acrosome, although is still formed, it does not appear to develop symmetrically and appears to detach from the nucleus in condensed spermatids. Another structure that is impaired in CCP5-KO spermatids in the manchette. Manchettal MTs, are seen to emanate from ectopic regions of the germ cells without running parallel to the nucleus, and are often observed within the spermatids nuclei. Altogether these defects correlate with an aberrant-shaped spermatid nucleus not showing the typical hook-like shape. Another phenotype observed in CCP5-KO elongating spermatids is the presence of supernumerary basal bodies that correlates with the presence of singlet or doublets microtubules dispersed within the germ cell cytoplasm. Interestingly sperm accessory structures are seen to chaotically organize around the microtubules. Unstable disassembling axonemes are seen together with those MTs, suggesting that CCP5-KO spermatids develop abortive unstable flagella. Interesting all these ultrastructural defects correlate with increased level of glutamylation on round spermatids’ cortical MTs and elongating spermatids’ manchettal MTs. Taken together, this study strongly suggests that CCP5-mediated glutamylation regulation is fundamental for spermatids differentiation into healthy functional spermatozoa.

In the fission yeast S. pombe, Mto1p and Mto2p are involved in MT nucleation from cytoplasmic MTOCs and recruit components of the γ-TuC. We wished to discover whether Ben1p plays a similar role. ben1+ was tagged at its normal chromosomal location and the intracellular localization was analyzed. It showed that Ben1p localized to cytoplasmic microtubules. We generated ben1Δ. cells by replacing it with a copy of ura4+. We observed that 20% of the cells were undergoing division. Next we observed the effects that deleting ben1+ would have on the functions of Mto1p and Mto2p. We setup crosses between these strains and observed their localization. We did not observe any specific Mto2-GFP localization in ben1Δ. cells. This suggests that Ben1p plays a role in the proper localization of a γ-TuC associated protein.

Cytokinesis separates replicated chromosomes and cytoplasm into two daughter cells. In animal cells, this is achieved by the formation of a cleavage furrow that bisects the mitotic (or meiotic) spindle. It is known that the mitotic apparatus defines the cell cleavage plane. However, it is not clear how the mitotic apparatus initiates the cleavage furrow. Each part of the mitotic apparatus; namely asters, central spindle (microtubule arrays and the spindle midzone), and chromosomes, has been found capable of inducing a cleavage furrow in certain cell types. Yet it is uncertain which part is the essential source of the signal and whether all parts act in concert. This thesis systematically examines in grasshopper spermatocytes 1) which spindle constituent is the essential source of furrow signal; 2) the impact of microtubules on distribution of actin filaments and positioning of cell cleavage relative to spindle reorganization; 3) the independent role of the spindle midzone relative to microtubules in furrow initiation and ingression. These examinations combine micromanipulation with digital-enhanced polarization microscopy and epifluorescence microscopy, in which mitotic spindles in living cells are mechanically dissected and rearranged as desired as well as microfixed to evaluate and propose models for cell cleavage. This thesis has come to the conclusion that none of structural constituents of the spindle apparatus is essential for cell cleavage induction except microtubules. First, furrow induction occurs regardless of a particular spindle constituent, so long as sufficient microtubules are present to form bipolar arrays. Second, microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage. Asymmetric alterations of spindle microtubules dynamically affect the location of the spindle midzone, distribution of actin filaments, and ultimately position of the cleavage furrow in cells containing a bipolar spindle, monopolar spindle, or half-spindle. Third, actin filaments are distributed to the furrow region by microtubule-mediated transport, but organized by the midzone, which is essential for furrow ingression, but not initiation. These results suggest that during post-anaphase spindle assembly, actin filaments are excluded by bipolar microtubule arrays to the equatorial cell cortex where they bundle into a contractile ring with cytokinetic factors. / Graduation date: 2004