HER2overexpression is associated with poor breast cancer prognosis and increased risk of metastasis. Current HER2targeted therapies include monoclonal antibody based strategies which work by reducing HER2 levels at the cell surface (trastuzumab), by preventing HER2 dimerization (pertuzumab), or via targeted delivery of a cytotoxic payload (trastuzumab emtansine). Although these therapies are successful in some cases, acquired and inherent resistance to these therapeutics remain a treatment hurdle. Oncolytic viruses (OVs) specifically target and lyse cancer cells while leaving normal cells unharmed. One such OV, VSVΔ51, replicates in interferon (IFN) defective cells, a characteristic of approximately 70% of tumours. We hypothesized that the combination of HER2 targeting therapies with VSVΔ51 could improve therapeutic efficacy. We found that HER2 overexpression was associated with increased virus sensitivity and that modulation of HER2 signaling through a subset of activating ligands and inhibitory drugs could influence infection. We further established that the HER2 monoclonal antibodies trastuzumab and pertuzumab mediate an anti-viral effect on VSVΔ51 spread. Finally, we demonstrate that conjugation of a microtubule targeting agent to trastuzumab can overcome the induced anti-viral state and enhance VSVΔ51 spread specifically in cancer cells. Overall, this work highlights the importance of HER2 signaling and activation on VSVΔ51 spread and shows that conjugation of microtubule destabilizing agents to monoclonal antibodies can enhance VSVΔ51 efficacy.

Le cancer du sein touche une femme sur neuf dans le monde et constitue un problème majeur de santé publique. L’identification de nouveaux biomarqueurs pour un traitement personnalisé pour les tumeurs du sein de plus mauvais pronostic, dites « triple-négatives », est extrêmement urgent. ATIP3, le produit majeur du gène candidat suppresseur de tumeurs MTUS1, a été identifié par l’équipe comme étant un biomarqueur des tumeurs du sein les plus agressives. De plus, ATIP3 inhibe la prolifération et la migration in vitro, ainsi que la progression tumorale et la formation de métastases in vivo et constitue une cible thérapeutique. ATIP3 est une protéine associée aux microtubules (MT) en interphase et au fuseau mitotique durant la mitose. Mon projet de thèse a pour objectif principal d’identifier les partenaires d’interaction d’ATIP3 impliqués dans ses mécanismes d’action antitumoraux. Dans une première partie, j’ai montré qu’ATIP3 interagit avec EB1, une protéine majeure de la dynamique du MT. L’interaction ATIP3-EB1 diminue l’accumulation d’EB1 à l’extrémité croissante du MT. Un nouveau mécanisme a été proposé dans lequel l’interaction ATIP3-EB1 réduit indirectement la vitesse d’échange d’EB1 à son site de liaison au bout plus du MT, ayant pour conséquence une diminution de la dynamique du MT. Dans une deuxième partie, j’ai montré qu’une déplétion d’ATIP3 induit une réduction de la taille du fuseau mitotique. Une analyse protéomique a permis d’identifier la kinésine Kif2A comme partenaire d’interaction d’ATIP3. ATIP3 forme un complexe avec Kif2A et Dda3 qui est dépendant d’une phosphorylation par Aurora kinase A. ATIP3 maintient la taille du fuseau en diminuant le recrutement de Kif2A et Dda3 au pôle de façon dépendante d’AurKA. ATIP3 régule donc négativement ses partenaires d’interaction. Enfin, dans une troisième partie, la relevance clinique du couple ATIP3-EB1 a été évaluée et j’ai montré que l’expression combinée des deux biomarqueurs ATIP3 et EB1 était associée à l’agressivité de la tumeur et à une survie diminuée. Ainsi, l’ensemble de mes travaux a permis de mettre en évidence de nouvelles cibles thérapeutiques afin de mettre en place des traitements personnalisés / Breast cancer is a leading cause of death by malignancy in women worldwide. The identification of new molecular markers for personalized treatment of poor prognosis breast tumors, such as those of the triple negative subtype, is urgently needed. Our team is leader in the study of ATIP3 protein, encoded by candidate tumor suppressor gene MTUS1. ATIP3 is down-regulated in 85% of triple negative breast tumors, and low levels of ATIP3 are associated with poor survival of the patients. We have shown that ATIP3 reduces proliferation and migration in vitro, and tumor growth and metastasis formation in vivo. ATIP3 localizes along the microtubule (MT) in interphase and on the mitotic spindle and spindle poles during mitosis. My PhD project aimed at identifying ATIP3 partners involved in its anti-tumoral effects. In the first part, I will present data showing that ATIP3 interacts with EB1, a major regulator of MT dynamics. ATIP3-EB1 interaction prevents EB1 accumulation at MT growing ends. I proposed a novel mechanism by which ATIP3-EB1 indirectly reduces EB1 turnover at its binding site at MT plus end, which consequently reduces MT dynamics. In the second part of my thesis, I showed that ATIP3 silencing induces reduced spindle length. In parallel, I identified the MT-depolymerizing kinesin Kif2A as an ATIP3 partner by proteomic analysis. ATIP3 forms a complex with Kif2A and Dda3 in an AurKA-dependent manner. I showed that ATIP3 maintains mitotic spindle size by inhibiting Kif2A and Dda3 recruitment at the spindle pole. My study also revealed a recriprocal regulation between ATIP3 and AurKA. Thus, ATIP3 negatively regulates its binding partners. Finally, in a third part, clinical relevance of ATIP3-EB1 in breast cancer has been evaluated and I showed that combinatorial expression of ATIP3 and EB1 is associated with tumor agressiveness and reduced patient survival. Altogether, this work highlighted new therapeutic targets to propose personalized treatments.

La mise en conformation de l’α et β tubulines en hétérodimeres polymérisables nécessite l’intervention de cinq protéines « Tubulin Binding Cofactors » (TBCA a TBCE) dont TBCC qui joue un rôle indispensable. Dans des cellules humaines d’adénocarcinome mammaire, nous avons modifié le niveau d’expression de TBCC et nous avons montre que ceci avait un impact sur le contenu des fractions de tubuline, la dynamique des microtubules ainsi que sur le phénotype et chimiosensibilité des cellules. La distribution en cycle cellulaire et les durées de la mitose et de la phase S ont été altérées. La modification de TBCC avait un faible effet sur la vitesse de prolifération in vitro par contre les cellules présentaient des différences significatives de croissance tumorale in vivo. Les réponses aux agents antimicrotubulaires et à la gemcitabine ont montrées une chimiosensibilité dépendante de la distribution en cycle cellulaire. Tous ces résultats montrent l’importance de la régulation du contenu en tubulines et l’impact de ceci sur le comportement de la cellule en général et vis-à-vis des traitements / The proper folding pathway of α and β-tubulin into the α/β-tubulin heterodimers involve five Tubulin Binding Cofactors (TBCA to TBCE). TBCC plays a crucial role in the formation of polymerization-competent the α/β-tubulin heterodimers. To evaluate the impact of microtubule mass and dynamics on the phenotype and chemosensitivity of breast cancer cells, we targeted TBCC in human breast adenocarcinoma and developed variants of breast cancer cells with modified content of TBCC. We have shown that the modifications in TBCC expression level influenced tubulin fraction distribution and microtubule dynamics. Cell cycle distribution and the durations of mitosis and S-phase were altered. The proliferation rate in vitro was slightly modified whereas in vivo the TBCC variants presented major differences in tumor growth capacity. Chemosensitivity to antimicrotubule agents (paclitaxel and vinorelbine) as well as to gemcitabine was observed to be dependent on the cell cycle distribution of the TBCC variants. These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype, cell cycle progression and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterization of resistant phenotypes

Many active processes in cells are driven by highly specialised motor proteins, which interact with the cytoskeleton: a network of filamentous structures, e.~g.~ actin filaments and microtubules, which organises intracellular transport and largely determines the cell shape. These motor proteins are able to transduce the chemical energy, stored in ATP molecules, to do mechanical work while interacting with a filament. Certain motor proteins, e.~g.~members of the KIN-13 kinesin subfamily, are able to interact specifically with filament ends and induce depolymerisation of the filament ends. One important role for KIN-13 family members is in the mitotic spindle, a microtubule structure that is formed in the process of cell division and is responsible for separation and distribution of the duplicated genetic material to the forming daughter cells. The aim of this work is to develop a theoretical framework capable of describing experimentally observed behaviour and shed light on underlying principles of motor induced filament depolymerisation. We use two different theoretical approaches to describe motor dynamics in this non- equilibrium situation: On the one hand we use phenomenological continuum equations which themselves are to a large extent independent of the underlying molecular details of the system. Molecular details of the system are incorporated in the equations through the specific values of macroscopic parameters which are determined by the underlying details. On the other hand, we use one- and two-dimensional discrete stochastic descriptions of motors on a filament. These kind of descriptions enable us to investigate the effects of different microscopic mechanisms of filament depolymerisation, and to investigate the role of fluctuations on the dynamic behaviour of motor proteins. We additionally discuss filament depolymerisation in the case where motors are not free to move but are fixed to a common anchoring point and depolymerise filaments under the influence of applied forces, mimicking the situation in the mitotic spindle. Our results can be related to recent experiments on members of the KIN-13 subfamily and predictions made in our theory can be tested by further experiments. Although motivated by experiments involving members of the KIN-13 subfamily, our theory is not restricted to these motors but applies in general to associated proteins which regulate dynamics of filament ends.

Oesophageal cancer is a malignant and aggressive disease with very poor survival. The aim of this thesis was to evaluate novel therapeutic targets in oesophageal cancer. In paper I, Hsp90 was expressed in all 81 oesophageal cancer tissues and also in nine oesophageal cancer cell lines. A specific Hsp90 inhibitor, 17-AAG, could efficiently inhibit cell proliferation, cell survival and sensitise oesophageal cancer cells to gamma photon irradiation. By inhibition of Hsp90 using 17-AAG, EGFR- and IGF-1R-mediated signalling was downregulated. In paper II, tumour samples from 80 oesophageal cancer patients were investigated for the expression of EGFR and LRIG1-3. Based on a total score of intensity and expression fraction a trend towards survival differences was found for LRIG2 (p=0.18) and EGFR (p=0.09). Correlation analysis revealed a correlation between expression of EGFR and LRIG3 (p=0.0007). Significant correlations were found between LRIG1 mRNA expression levels and sensitivity to cisplatin (r = –0.74), docetaxel (r = –0.69), and vinorelbine (r = –0.82). In paper III, microtubule targeting drugs podophyllotoxin (PPT), vincristine and docetaxel inhibited survival and proliferation of oesophageal cancer cells. Unexpectedly, experiments showed that microtubule destabilising agents inhibited EGFR phosphorylation and signalling. A tyrosine phosphatase inhibitor, sodium orthovanadate, was able to reverse the EGFR dephosphorylation. In paper IV, imetelstat, a telomerase antagonist, inhibited telomerase activity, colony formation ability and decreased proliferation of oesophageal cancer cells. Inhibition of telomerase activity by imetelstat led to an increase of 53BP1 foci indicating induction of DSBs. Furthermore, the fraction and size of radiation-induced 53BP1 foci were increased by imetelstat pre-treatment. In conclusion, Hsp90 and telomerase represent potential therapeutic targets in oesophageal cancer. And, the implication of EGFR and LRIG as prognostic factors is limited. Furthermore, disruption of the microtubule network may activate a protein tyrosine phosphatase that can regulate EGFR phosphorylation.

B. Cinerea, l'agent responsable de la pourriture grise, possède deux alpha-tubulines : l'alpha-tubuline A et l'alpha-tubuline B. Ces protéines sont connues pour participer à la construction des microtubules, mais la connaissance de leurs rôles est relativement limitée chez les champignons et particulièrement chez les pathogènes des plantes. L'analyse du profil d'expression des gènes codant pour ces tubulines a démontré que l'alpha-tubuline B est majoritairement exprimé et qu'il se caractérise par un pic au moment de la germination de la spore fongique. L'étude du promoteur de son gène a révélé la présence de trois zones possibles de régulation, et l'approche de «simple-hybride» a permis de mettre en évidence deux régulateurs potentiels : BcYOH1 et BcFT027. Un mini-réseau de régulation entre les différents gènes codants pour les tubulines et ces facteurs de transcription a été mis en évidence. Nos résultats ont montré que le gène codant pour l'alpha-tubuline B n'est pas indispensable à la vie cellulaire et l'étude du mutant de délétion a permis d'attribuer un rôle particulier de cette protéine dans la formation du mycélium aérien et, donc dans la production des spores de la reproduction asexuée, mais également dans le cycle infectieux du champignon. Par ailleurs, les mutants de délétion des gènes codant pour les deux facteurs de transcription identifiés dans notre étude ont permis l'analyse de leur rôle sur le développement et le pouvoir infectieux du champignon. Nous avons ainsi montré que la vitesse de croissance, la biomasse, le mycélium aérien et la vitesse d'infection étaient augmentés chez le mutant BcFT027 / The plant pathogen Botrytis cinerea contains two alpha-tubulin isomers (alpha-tubulin A and B). These proteins are known to participate in the construction of microtubules in all eukaryotes, but our knowledge about the roles of different isomers is particularly poor in fungi, and null in fungal plant pathogens. Analysis of gene expression profiles in B. cinerea revealed that the alpha-tubulin B encoding gene is more expressed than the alpha-tubulin A one and that its expression peaks during spore germination. Subsequent promoter studies led to the identification of three DNA regions probably involved in this regulation, and two putative regulators were then found by using the one-hybrid yeast system : the already discovered BcYOH1 and the totally unknown and ascomycetous specific BcFT027. Additional expression studies in mutant strains of these regulators finally suggested the existence of a regulatory network between these two regulators and the two alpha-tubulin encoding genes. Production and analysis of alpha-tubulin B deletion mutants showed that this isomer is not essential for cell viability in B. cinerea. More importantly, this study revealed that the alpha-tubulin protein plays a role during plant infection as well as in the formation of aerial mycelium and the production of asexual spores. Partial to complete characterization of the BcYOH1 and BcFT027 deletion mutants strengthened these results and showed that BcFT027 is a key player in the development of areal mycelium and of the infection process (via the development of penetration structures called infection cushions). Never reported before, these results are of significant interest in our understanding of tubulins and fungal development

<p> The urgency for deeper knowledge about nervous system function and dysfunction has never been greater. With increasing rates of mental disorders and expanding healthcare costs, deciphering details of axonal development is essential to meeting this imperative. Models of neuronal growth are improving as roles of microtubules and motor proteins surface. However, traditional motor protein studies focus on intracellular cargo transport, leaving deficits in knowledge about how these proteins organize cytoskeletal filaments in the axon and growth cone during neuronal development. Inconsistent findings on microtubule activity in growing axons also leave gaps in quantitative assessments of microtubule translocation and assembly, limiting the ability to construct a comprehensive model of axonal elongation.</p> The goal of this study was to provide a more complete neuronal growth cone model by determining how individual microtubule translocation and assembly, mass microtubule movements, and motor protein activity contribute to axonal elongation. The underlying mechanisms of these processes were investigated by testing the roles of dynein and microtubule dynamics in axonal elongation of <i>Aplysia </i><i>californica </i>neurons using transillumination, fluorescent speckle, and super-resolution microscopy. Pharmacologically inhibiting either dynein activity or microtubule assembly reduced both bulk and individual microtubule anterograde translocation and neurite elongation rates. Suppressing both processes simultaneously had compensatory rather than additive effects. Super-resolution imaging also revealed fewer dynein motors co-localized with microtubules when microtubule assembly was inhibited. These results strongly suggest that disrupting microtubule assembly blocks neurite outgrowth partly because it inhibits dynein-mediated bulk microtubule translocation.

The functionalization of the outer lattice of in vitro reconstituted microtubules has paved the way for the development of diverse nano-device applications. The outer lattice has been metallized for the bottom-up synthesis of nanowires composed of various materials. Moreover, a wide range of biomolecules and nanoprobes have been attached to the outer surface for nano-scale transport and detection assays in conjunction with motor proteins. The functionalization of the outer lattice has certain implications: While the nanowires adopt the overall shape of the microtubules, their surface is inhomogeneous due to the absence of any morphological control. The attachment of cargo on the outer lattice creates a ‘roadblock effect’ hindering the transport activity of the motor proteins as they share a common substrate surface. In this project, the utilization of the hollow interior region of the microtubules, called the lumen (∼15 nm in diameter) is proposed to overcome these limitations. A strategy is developed to functionalize the microtubule lumen by targeting molecular cargo conjugated to lumen-binding (anti-acetyl alpha-tubulin lysine-40) antibodies. This would optimize existing motility-based applications as the outer surface would be exclusively available for the activity of the motor proteins. Furthermore, microtubules functionalized with luminal gold nanoparticle ‘seeds’ are utilized for the lumen-templated assembly of gold nanowires.

No description available.

Endocytosis is essential for the pathogenic development of Ustilago maydis. It has been shown that the initiation of pathogenicity relies upon the ability of the cell to recognize pheromone (a1 or a2) released from its mating partner and subsequently to form conjugated hyphae. The actin and microtubule cytoskeleton plays an essential role in all aspects of cell growth. A component of the actin cytoskeleton, the filamentous actin is required for cell-cell fusion, whereas the molecular motors, kinesin and dynein, move along microtubules and provide the long distance transport of many proteins and they are important in cell growth and pathogenicity. In this thesis, we investigated the role of the cytoskeleton in endocytosis and a1 pheromone transport, using a fluorescently labelled derivative of the a1 pheromone. We confirmed that uptake of the a1 pheromone is also receptormediated. In addition, we have shown that pheromone transport towards the cellular vacuole requires the actin and microtubule cytoskeletons. Furthermore, we revealed that the microtubule-dependent motors kinesin-1 and kinesin-3 and dynein were shown to be essential in the delivery of the pheromone to vacuoles. Moreover, a mutation in the early endosomal protein Yup1 gene causes a stop in delivery of the synthetic pheromone to the vacuole. This suggests that it travels with early endosomes. Within the actin cytoskeleton, we analysed the dynamics of actin patches in the presence of the synthetic pheromone and found that the dynamics of the patches increased significantly. Additionally, in the presence of an over-expression of the tail domain of the molecular motor myosin-5, the dynamics of the patches were significantly reduced and their intensity diminished.