Chemical modification is a powerful technique for probing functionally important amino acids. N-ethylmaleimide (NEM) reacts readily with exposed sulfhydryl groups, and has previously been shown to inhibit the activity of MT-motor proteins and tubulin assembly. This project seeks to investigate the mechanisms by which NEM affects motor function and inhibits MT minus end assembly. Recombinant motor domains of Drosophila kinesin (DK350 and DK375), Ncd (MC1), and squid kinesin (p181) were modified by NEM. NEM treatment was shown to affect the binding of MC1, but not recombinant kinesin proteins to MTs in the co-sedimentation assay. NEM treatment decreased the MT-stimulated ATPase rates of MC1 and DK350 in an NEM-concentration dependent manner, but did not affect the rate of DK375. Observed effects with DK375, p181, and MC1 were correlated with the number of labeled cysteines determined with [3H]NEM. As previously known, when NEM-treated tubulin was combined with untreated tubulin at certain ratios, assembly occurred only at the MT plus end. To investigate the mechanism by which NEM affects the polarity of tubulin assembly, tubulin was treated with NEM and assembly was analyzed using video-enhanced differential interference contrast microscopy. [3H]NEM was used to follow the time course of modification and to determine the number of modified sites per tubulin subunit. After 10 minutes, one cysteine was labeled on both a and b tubulin and this was sufficient to inhibit minus end assembly. Additionally, having one subunit labeled out of five tubulin subunits was sufficient to observe this effect. Protein digestion methods were used to aid in elimination of cysteines, to characterize potential critical cysteines in MC1, a, and b tubulin. / Master of Science

Résumé Les protéines XMAP215/ch-TOG appartiennent à une famille de protéines associées aux microtubules (MAPs), bien conservée tout au long de l'évolution, la famille XMAP215/Dis1. Cette famille joue un rôle dans la régulation du cytosquelette des microtubules (MT), en particulier pendant la division cellulaire. Chez l'humain, ch-TOG est la protéine surexprimée dans les tumeurs du colon et du foie, une protéine qui provient de cellules blastiques et de plusieurs formes de cancer. Certaines protéines XMAP215/ch-TOG ont été retrouvées dans différentes localisations cellulaires, toujours reliées aux MTs, donnant origine à une activité spécifique. Cependant, la localisation exacte de XMAP215/ch-TOG ainsi que son activité restait à être déterminées. Dans ce contexte scientifique, nous avons développé une série d'anticorps monoclonaux (mcAB) qui nous ont permis d'identifier deux populations différentes de la famille des protéines XMAP215/Dis1. Les images de microscopie confocale des cellules fixées ont montré une première localisation, la colocalisation bien connue XMAP215-microtubulaire (MT-XMAP215) qui s'observe pendant l'interphase et pendant la mitose (fuseau mitotique). Une deuxième localisation a été identifiée sur le bout plus des MTs, donnant XMAP215/ch-TOG comme faisant parti de la famille des protéines de bout plus (+TIPs). Cette deuxième colocalisation a été identifiée comme +TIP XMAP215/ch-TOG. La +TIP XMAP215 est la protéine la plus distale du bout des MTs. La hiérarchie a été établie en faisant la comparaison de la localisation de XMAP215/ch-TOG avec les protéines les plus connues du bout plus, telles qu'EB1, CLIP170 et p150Glued. Dans l'extrait mitotique de Xenopus laevis, les images obtenues in vivo par la microscopie de fluorescence par réflexion totale interne (TIRF) ont permis d'identifier une +TIP XMAP215 présente au bout des MTs qui polymérisent et dépolymérisent. Les images de microscopie cryo-électronique (Cryo-EM) ont montré une activité spécifique de la population +TIP XMAP215. Dans les solutions de tubuline pure, XMAP215 induit la formation de structures au bout des MTs, cette activité est compatible avec les mécanismes de croissance des MTs. Sur la base de nos résultats, nous proposons un modèle où XMAP215 se charge des dimères de tubuline en devenant une structure de type protofilament. Cette structure se lie au bout du MT en utilisant son domaine C-terminal, en rajoutant les dimères de tubuline et aussi certainement en participant à la fermeture de la structure microtubulaire même. La protéine interviendrait donc dans la dépolymérisation et aurait un rôle dans le mécanisme de dépolymérisation contrôlée. Une fois que l'addition de tubuline a eu lieu, la +TIP XMAP215 pourrait évoluer en MT-XMAP215, la forme la plus connue de la protéine qui a été associée au trafic des granules d'ARN. Mots cles : XMAP215, ch-TOG, microtubules, anticorps monoclonaux. / Summary XMAP215/ch-TOG are members of an evolutionary conserved family of microtubule-associated proteins (MAPs), the XMAP215/Dis1 family. This family of proteins plays a key role in the regulation of the microtubule (MT) cytoskeleton, particularly during the cell division. In humans, ch-TOG is the colon-hepatic tumor overexpresed gene, a protein whose sequence was originally reported from blastic cells and from several forms of cancer. A few members of the XMAP215/ch-TOG family have been found to be present in different cell localizations, always MT-related, perhaps providing in this way a selected activity. However, the XMAP215/ch-TOG exact localization and activity has remained as a theory to be probed. In this scientifical context, we developed a series of monoclonal antibodies (mcABs) that allow us to identify two different populations of the XMAP215/ Dis1 family of proteins. Confocal images of fixed cells revealed a first and well known XMAP215/ch-TOG population, a microtubular-XMAP215 (MT-XMAP215), co-localizing with microtubules (MTs) in interphase and mitotic spindle. A second localization was identified at the tips of growing MTs, placing XMAP215/ch-TOG as one more member of the known microtubule plus end tracking proteins (+TIPs). This second population was identified as the +TIP XMAP215/ch-TOG. The + TIP XMAP215 is the most distal +TIP protein at the tip of MTs. The + TIPs hierarchy was established comparing the XMAP215/ch-TOG localization with other well known + TIPs proteins such as EB1, CLIP170 and p150Glued. In the Xenopus laevis mitotic egg extract, the Total Internal Reflection Fluorescence (TIRF) In vivo images, identified a +TIP XMAP215 that is present at the tip of polymerizing and depolymerising MTs. Cryo-electron microscopy (Cryo-EM) images probed a selective activity for the +TIP XMAP215 population. In pure tubulin solutions XMAP215 shown to promote the formation of outwardly sheet structures at the tip of MTs, compatible with growing mechanisms in MTs. Based in our results we propose a model where free XMAP215 is previously loaded with tubulin dimers to become a protofilament-like structure. This structure joins the MT tip using its C-terminal domain, not only adding the tubulin dimmers but also perhaps participating in the MT sheet closure. The possibility that the protein participates in the MT depolymerization could be associated to a “controlled” depolymerization mechanism. Once the tubulin addition has taken place, the +TIP XMAP215 protein could evolve to a MT-XMAP215, the well know form of the protein that has been related to the RNA granules traffic. Key words: XMAP215, ch-TOG, microtubules, monoclonal antibodies.

La mitose est une étape essentielle du cycle cellulaire à l’issue de laquelle le génome répliqué de la cellule mère est ségrégé de façon équitable entre les deux cellules filles. Pour cela, la cellule assemble une structure hautement dynamique et composée de microtubules, appelée le fuseau mitotique. En plus d’assurer la bonne ségrégation des chromosomes, le fuseau mitotique détermine l’axe de division, un phénomène particulièrement important pour la division asymétrique où des déterminants d’identité cellulaire doivent être distribués de façon inéquitable entre les deux cellules filles. L’assemblage et la dynamique de ce fuseau sont finement régulés par de nombreuses protéines qui sont associées aux microtubules. Au cour de ma thèse, nous avons identifié 855 protéines constituant l’interactome des microtubules de l’embryon de Drosophile par spectrométrie de masse puis criblé par ARNi 96 gènes peu caractérisés pour un rôle en mitose dans le système nerveux central larvaire. Par cette approche, nous avons identifié 18 candidats sur la base de leur interaction aux microtubules et de leur phénotype mitotique, dont Ensconsine/MAP7. Nous avons montré qu’Ensconsine est capable de s’associer aux microtubules du fuseau et favorise leur polymérisation. De plus, les neuroblastes des larves mutantes présentent des fuseaux raccourcis et une durée de mitose prolongée. Ce délai en mitose est dû à une activation prolongée du point de contrôle du fuseau mitotique qui est essentiel pour une ségrégation correcte des chromosomes en l’absence d’Ensconsine. D’autres part, en association avec la Kinésine-1, son partenaire fonctionnel en interphase, nous avons montré qu’Ensconsine est également impliquée dans la séparation des centrosomes au cours de l’interphase. Ceci entraine une distribution aléatoire des centrosomes pères et fils dans cellules filles. Grâce à cette étude, nous avons révélé deux nouvelles fonctions pour Ensconsine : elle favorise la polymérisation des microtubules et participe donc à l’assemblage du fuseau mitotique et est impliquée, avec la Kinésine-1 dans la dynamique des centrosomes. / Mitosis is a key step of the cell cycle that allows the mother cell to segregate its replicated genome equally into the two daughter cells. To do so, the cell assembles a highly dynamic structure composed of microtubules called the mitotic spindle. Additionally to its role in the faithful segregation of chromosomes, the mitotic spindle defines the axis of cell division. This phenomenon is particularly important for the asymmetric cell division in which cell fate determinants have to be unequally distributed between the two daughter cells. Spindle assembly and dynamics are subtly regulated by numerous microtubules-associated proteins. During my PhD, we identified using mass spectrometry, 855 proteins establishing the Drosophila embryo microtubule interactome. An RNAi screen was performed in the larval central nervous system for 96 poorly described genes, in order to identify new mitotic regulators. Based on microtubule interaction and mitotic phenotype, among 18 candidates we focused on Ensconsin/MAP7. We have shown that Ensconsin is associated with spindle microtubules and promotes their polymerization. Neuroblasts from mutant larvae display shorter spindles and a longer mitosis duration. This mitotic delay is a consequence of an extended activation of the spindle assembly checkpoint, which is essential for the proper chromosome segregation in the absence of Ensconsin. This study also showed that, in association with its interphase partner Kinesin-1, Ensconsin is involved in centrosome separation during interphase. As a result, mother and daughter centrosomes are randomly distributed between the daughter cells. In conclusion, we highlighted two news functions of Ensconsin : first, this protein promotes microtubule polymerization and is involved in spindle assembly ; second, Ensconsin and its partner Kinesin-1 regulate centrosome dynamics.

Les agents pharmacologiques ciblant la dynamique des microtubules (MTs) sont très utilisés en chimiothérapie des cancers agressifs. Le paclitaxel (PTX) est utilisé depuis des décennies et donne de bons résultats pour le traitement des tumeurs solides. Plusieurs inconvénients, notamment ses effets secondaires et la résistance de certains cancers limitent cependant l'efficacité de ce médicament. Dans le but d'identifier de nouveaux composés pharmacologiques qui sensibilisent les cellules au PTX, nous avons recherché, parmi une collection de 8000 molécules, celles capables de sensibiliser des cellules cancéreuses à une dose non toxique de PTX. Nous avons ainsi sélectionné un composé de la famille des carbazoles : Carba1. Dans les cellules, l’association carba1/PTX a un effet cytotoxique supérieur à la somme des effets de carba1 et de PTX, quand ces molécules sont appliquées séparément, indiquant un effet synergique. De plus, des analyses approfondies de différents phénotypes ont permis de montrer que l'administration de carba1 avait pour conséquence d'amplifier des effets du PTX.À fortes doses, carba1 entraine un blocage des cellules en prométaphase mais n’altère pas le réseau microtubulaire, ni en interphase ni en mitose. En revanche, in vitro, carba1 cible la tubuline en se fixant sur le site colchicine, provoquant un retard et une diminution de la polymérisation des MTs. En plus de la tubuline, des études génétiques réalisées sur la levure suggèrent que carba1 a d'autres cibles dont CENP-E, kinésine essentielle à l’alignement des chromosomes au cours de la mitose.Des études menées sur un modèle de cancer mammaire murin agressif (allogreffes) ont révélé que carba1 seul et carba1/PTX ne présentaient aucune toxicité. De plus, les effets anti-tumoraux et anti-métastatiques de la combinaison carba1/PTX sur ces modèles se sont montrés encourageants, bien que des mises au point, notamment sur la posologie sont encore à prévoir. Carba1 est une molécule nouvelle, avec des applications jusque-là inconnues. C’est pourquoi une déclaration d’invention, en vue d'un dépôt de brevet, a été soumise au CNRS. / Microtubules (MTs) targeting agents are a powerful weapon in the war against aggressive cancers. Paclitaxel (PTX) has been used successfully for the treatment of solid tumors for decades. Several features, including side-effects and resistance of some cancers make this drug not always effective. With the aim to identify new chemical compounds that sensitize cells to paclitaxel we screened a library of 8,000 compounds, to select those not toxic for cell cultures when applied alone, that become toxic when applied in combination with a non-toxic dose of paclitaxel. This lead to the selection of a carbazole derivative: carba1. In cells, the carba1/PTX combination has a greater cytotoxic effect than the addition of the effects of each drug assayed separately, indicating a synergistic effect. In addition, in-depth phenotypic analyzes indicate that the administration of carba1 amplify the effects of PTX.High doses of carba1 induce a cell blockade in prometaphase, but do not alter the MT network in interphase or mitosis. In contrast, in vitro, carba1 targets the tubulin colchicine binding site, causing a delay and a decrease in MT polymerization. Genetic studies conducted on yeast indicated other potential additional targets including CENP-E, an essential kinesin for chromosome alignment during mitosis.Studies conducted on a preclinical mouse model of aggressive breast cancer (orthotopic grafts) revealed that carba1 alone and carba1/PTX showed no toxicity. In addition, the anti-tumor and anti-metastatic effects of the carba1/PTX combination on these models have been encouraging, but an optimization of the posology is still needed. Carba1 is a new molecule, with previously unknown applications. This is why a declaration of invention, with a view to filing a patent, has been submitted to the CNRS.

No description available.

No description available.

Meckel-Gruber syndrome (MKS) is a recessive genetic disease that is uniformly lethal in affected children due to resultant developmental defects in the kidney and brain. 13 MKS genes have been identified, and further candidate genes have been linked to this disease, all encoding unrelated proteins. Their role is believed to be in generation and compartmentalisation of the primary cilium, a microtubule-based organelle that functions in signal transduction of developmentally-crucial pathways. However, recent evidence indicates that these proteins are also likely involved in regulation of the actin cytoskeleton. Furthermore, research is beginning to uncover roles of other ciliopathy proteins in regulation of additional subcellular structures, such as the microtubule cytoskeleton, focal adhesions and the Golgi. To begin to understand the roles of the MKS proteins beyond the cilium, I examined a number of cellular features of patient fibroblasts carrying mutations in TMEM216 (MKS2) and TMEM67 (MKS3). In this thesis, I describe the temporal appearance and nature of prominent actin bundles observed in these cells, and analyse the dependency of these on the Rho/ROCK signalling pathway. Furthermore, I identify novel alterations to the microtubule cytoskeleton and organisation of the Golgi complex in MKS patient cells, and subsequently establish a temporal order of these phenotypes, demonstrating microtubule defects as the first to occur in these cells. Finally, I connect these phenotypic defects to Rho/ROCK signalling. In contrast to the prevailing view in the ciliopathy field, I believe that a diffusion barrier at the transition zone is not the primary role of MKS proteins. Instead I propose, supported by these data, that MKS protein complexes play a dual role as effectors of Rho signalling in addition to performing a structural role with particular importance in tethering the cytoskeleton to membranes. I therefore conclude that these, and other ciliopathy protein complexes, may act as important signal transduction and structural components at multiple locations throughout the cell.

Les protéines associées aux microtubules (MAP) de structures, telles que celles appartenant à la famille des MAP1 sont connues pour contrôler la stabilité et la dynamique des microtubules (MTs). Elles sont aussi connues pour interagir avec des protéines post-synaptiques telles que les récepteurs GABAergique ou glutamatergique. Cependant, leur rôle pré-synaptique dans la libération de neurotransmetteurs a été très peu étudié. Dans cette thèse, j'utilise l'avantage du modèle Drosophila melanogaster dans lequel il n'y a qu'un seul homologue des MAP1 des vertébrés, nommé Futsch. J'ai étudié la fonction de Futsch à la jonction neuromusculaire (JNM) de larve, où cette protéine n'est trouvée que dans la partie pré-synaptique. Ici, j'ai montré qu'en plus de sa fonction connue sur la morphologie de la JNM (Roos et al., 2000; Gogel et al., 2006), Futsch est également important pour la physiologie de la JNM, par le contrôle de la libération de neurotransmetteurs ainsi que de la densité des zones actives (ZAs). J'ai montré que l'effet physiologique de Futsch n'est pas la conséquence de l'altération du cytosquelette de MTs ou d'un défaut de transport axonal, mais doit être la conséquence d'un effet local de Futsch à la terminaison synaptique. J'ai utilisé la microscopie d'éclairage structuré 3D (3D-SIM) pour étudier plus précisément la localisation de Futsch et des MTs au niveau de la ZA. Futsch et les MTs se trouvent presque toujours à proximité des ZAs, avec Futsch en position intermédiaire entre les MTs et les ZAs. En utilisant la technique de « proximity ligation assays », j'ai aussi démontré la proximité fonctionnelle de Futsch avec Bruchpilot un composant de la ZA, ce qui n'est pas le cas des MTs. En conclusion, mes données sont en faveur d'un modèle pour lequel Futsch stabilise localement les ZAs, en renforçant leur lien avec le cytosquelette de MTs sous-jacent. / Structural microtubule associated proteins like those belonging to the MAP1 family are known to control the stability and dynamics of microtubules (MTs). They are also known to interact with postsynaptic proteins like GABA or glutamate receptors. However, their presynaptic role in neurotransmitter release was barely studied. Here, we took advantage of the Drosophila model in which there is only one MAP1 homologue, called Futsch. We studied the function of Futsch at the larval neuromuscular junction (NMJ), where this protein is found presynaptically only. Here, we show that, in addition to its known function on NMJ morphology (Roos et al., 2000; Gogel et al., 2006), Futsch is also important for NMJ physiology, by controlling neurotransmitter release as well as active zone density. We show that this physiological effect of Futsch is not the consequence of disrupted microtubule bundle and disrupted axonal transport, but must be the consequence of a local effect of Futsch at the synaptic terminal. We used 3D-Structured Illumination Microscopy (3D-SIM) to further study the localization of Futsch and MTs with respect to active zones. Both Futsch and MTs are almost systematically present in close proximity active zones, with Futsch being localized in-between MTs and active zones. Using proximity ligation assays, we further demonstrated the functional proximity of Futsch, but not MTs, with the active zone component Bruchpilot. Altogether our data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying MT cytoskeleton.

Microtubules (MTs) are important components of the eukaryotic cytoskeleton, with critical functions in intracellular trafficking, establishing and maintaining cell morphology, and segregating chromosomes during mitosis. MTs are hollow, cylindrical polymers composed of αβ-tubulin heterodimers. The longitudinal assembly of αβ-tubulin subunits generates protofilaments, and multiple protofilaments (typically 13 in vivo) interact laterally to form the wall of the MT. In vitro, the polymerization of MTs proceeds in two steps: nucleation and elongation. During the nucleation phase, several αβ-tubulin subunits associate to form a seed, from which further MT elongation then occurs. However, at the relatively low αβ-tubulin concentrations found in vivo, the spontaneous assembly of MTs is not favoured, due largely to the slow kinetics of MT nucleation. The nucleation of MTs in vivo requires the γ-tubulin complex (γ-TuC), a ring-like complex composed of γ-tubulin and γ-tubulin complex proteins (GCPs). Two copies of γ- tubulin associate with one copy each of GCP2 and GCP3 to produce the γ-tubulin small complex (γ-TuSC). Multiple γ-TuSCs, along with the additional GCPs 4,5, and 6, assemble to form the larger γ-tubulin ring complex (γ-TuRC). The γ-TuRC contains a ring of 13 γ-tubulins, which acts as a template for the nucleation of MTs. Typically, the γ-TuC nucleates MTs only when localized to specific subcellular sites, referred to as microtubule organizing centres (MTOCs). However, the precise mechanism by which the γ-TuC is activated at MTOCs remains unknown. In fission yeast, the proteins Mto1 and Mto2 form a complex (Mto1/2) required for the nucleation and organization of cytoplasmic MTs. Mto1/2 determines sites of MT nucleation by recruiting the γ-TuC to several different MTOCs. Different sequences in the Mto1 C-terminus independently confer γ-TuC localization to spindle pole bodies, MTs, and the cell equator. Here, I show that the Mto1 N-terminus is necessary for localization to the nuclear envelope (NE). By simultaneously removing the N- and C-terminal localization domains, I generated the "Mto1-bonsai" mutant, which fails to localize to any conventional MTOCs. In mto1-bonsai cells, MTs are still nucleated in the cytoplasm in an Mto1- dependent manner, but nucleation is spatially random. This reveals that targeting of the γ- TuC to conventional MTOCs is not necessary for MT nucleation, and suggests that Mto1/2 has a direct role in activating MT nucleation by the γ-TuC. Live-cell confocal microscopy allows us to detect individual MT nucleation events, in which newly nucleated MTs are associated with single γ-TuCs as well as Mto1/2-bonsai complexes. Fluorescence quantification reveals that these nucleating complexes contain approximately 13 molecules of both Mto1-bonsai and Mto2, matching the 13 copies of γ-tubulin anticipated for a single γ-TuC. We propose that Mto1/2 may contribute to γ-TuC activation by promoting γ-TuSC assembly and/or inducing conformational changes in the γ-TuC upon binding. I also expressed and purified recombinant Mto1/2-bonsai complex, using a baculovirus/insect cell system. This recombinant Mto1/2-bonsai self-assembles into higher-order complexes, comparable in size to the complexes analyzed in vivo by fluorescence microscopy.

No description available.