Chapter 1 provides some background information on the disease area of cancer and current modes of treatment. Chemotherapy treatment is discussed with a focus on the major cellular targets for these drugs: DNA and microtubules. For each target, classes of active compounds are described along with their mode of action. The microtubule stabilizing agent laulimalide is introduced and a case is made for analogue synthesis. Chapter 2 describes our first generation efforts toward synthesis of des-methyl laulimalide. The target compound is divided into a northern and southern fragment with the synthesis of each fragment described. The preparation of landmark intermediates along the synthetic route is also described. Chapter 3 presents our ongoing efforts toward a second generation synthesis of des-methyl laulimalide. Our progress toward a des-methyl,des-pyran analogue is covered as well. Chapter 4 reviews progress in the area of laulimalide analogue development. Biological assay results and the first insights into structure-activity-relationships are described.

Factor Quinolinone Inhibitor 1 (FQI1), discovered as a small molecule inhibitor of the transcription factor LSF, causes cell death in many cancer cell lines and inhibits tumor growth in tumor xenografts and an endogenous hepatocellular carcinoma model in mice. Significantly, multiple animal studies have shown minimal to no toxicity after FQI1 treatment, making it a promising potential lead chemotherapeutic for multiple cancer types. In determining how FQI1 causes cancer cell death, it was previously shown that FQI1 treatment, like knockdown of LSF expression by siRNA, produced a mitotic arrest with condensed but unaligned chromosomes, but with no clearly observable transcriptional dysregulation. In this thesis, I establish that introducing FQI1 to cells already in mitosis induces a mitotic arrest in colorectal cancer cells, demonstrating that FQI1 inhibits mitotic processes directly while these processes are occurring. This mitotic arrest is characterized by defects in the mitotic spindle and limited connections of mitotic spindles to the kinetochores, as indicated by a dramatic decrease cold-stable microtubules in mitosis. Additionally, in a dose-dependent manner, FQI1 treatment resulted in supernumerary γ-tubulin-containing mitotic centrosomes and γ-tubulin-deficient aster-like bodies, indicating a defect in centrosome stability. As FQI1 is known to be a specific inhibitor of LSF, with its dose dependence for LSF inhibition directly proportional to its ability to inhibit cell proliferation, these findings suggested the novel hypothesis that LSF regulates mitosis through non-transcriptional mechanisms by interacting with key mitotic proteins required for proper spindle formation and metaphase alignment. By mass spectrometry, multiple proteins were identified that interact with biotinylated LSF in mitosis in a FQI1-sensitive manner, with several related to the formation and stability of the mitotic spindle. Proximity ligation assays validated endogenous LSF interactions with CKAP5, a processive microtubule polymerase that protects kinetochore microtubules from depolymerization, and MISP, a requirement for proper mitotic spindle positioning. However, in this assay these interactions were not demonstrably FQI1-sensitive. In conclusion, FQI1 treatment results in defects in kinetochore-microtubule attachment and centrosome stability, triggering a mitotic arrest. Combined with the target specificity of FQI1, this suggests the hypothesis that LSF is required for proper mitotic spindle formation through its protein interactions in mitosis. / 2022-10-16T00:00:00Z

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Using statistical physics principles to solve problems in biology is one of the most promising directions due to the complexity and non-equilibrium fluctuations in biological systems. In this work, we try to describe the dynamics at both cellular and molecular levels. Microtubule dynamics and dynamic disorder of enzyme proteins are two of the examples we investigated. The dynamics of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. However, critical discrepancies between experimental observations and existing models need to be resolved before further progress towards a complete model can be made. We carried out computational studies to compare the mechanical properties of two alternative models, one corresponding to the existing, conventional model, and the other considering an additional type of tubulin lateral interaction described in a cryo-EM structure of a proposed trapped intermediate in the microtubule assembly process. Our work indicates that a class of sheet structures is transiently trapped as an intermediate during the assembly process in physiological conditions. In the second part of the work, we analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can serve as a basic functional motif with properties normally discussed with larger networks in the field of systems biology. The work on slow enzyme dynamics fills the missing gap between studies on intramolecular and network dynamics. We also showed that enzyme fluctuations could be amplified into fluctuations in phosphorylation networks. This can be used as a novel biochemical "reporter" for measuring single enzyme conformational fluctuation rates. / Ph. D.

Mitosis, or cell division, is the mechanism by which cells divide and is an intricate process requiring the action and control of numerous proteins. Such proteins serve either as structural entities within the mitotic spindle, or perform the "work" within the apparatus. In particular, Kinesin-5 motor proteins, a subset within the kinesin motor protein superfamily, are primarily responsible for organization of microtubules (MTs) within the mitotic apparatus, and are consequently vital for efficient mitosis. These proteins utilize energy from ATP hydrolysis in order to "walk" along antiparallel MTs, positioning them into the bipolar mitotic spindle. Loss of Kinesin-5 activity results in formation of a monoastral spindle and subsequent cell cycle arrest. Recently, a wide variety of small molecules have been identified that possess the ability to inhibit certain Kinesin-5 motors. Such compounds, including monastrol (the first Kinesin-5 inhibitor identified), have been employed to study Kinesin-5 activity. A thorough understanding of Kinesin-5 function, combined with the ability to specifically target these proteins with small molecules, may provide the capability to control cell division and may therefore have significant implications in anti-cancer therapies. The following dissertation describes research that utilizes small molecules to probe the function (ATPase activity and MT interactions) of various Kinesin-5 proteins and provides information that will lead to a better understanding of exactly how such proteins function in vivo. Further, a greater knowledge of Kinesin-5 protein activity as well as specific interactions with small-molecule compounds, may lead to the development of more potent, less toxic anti-cancer drugs. / Ph. D.

Gastrulation, literally “formation of the gut” is ultimately an inadequate term to describe one of the most dynamic periods during vertebrate developmental biology. During gastrulation coordinated cell movements reshape the non-descript blastula into the structured gastrula and simultaneously specify the three germ layers: endoderm, mesoderm and ectoderm. The morphogenetic movements of gastrulation are highly conserved between species, but the links between their genetic and biomechanical regulation are poorly understood. The zebrafish embryo – externally hatched, optically clear and amenable to genetic manipulation – is an ideal vertebrate model in which to study both morphogenetic movements and their genetic control. This thesis provides a detailed analysis of the zebrafish Mix-type homeobox transcription factor, Mtx2, both in terms of its role in gastrulation and the molecular mechanisms regulated by Mtx2. This approach involved detailed examination of the Mtx2 loss-of-function phenotype and, subsequently, use of this phenotype as the basis for a microarray screen to identify and investigate Mtx2-dependent genes. One specific Mtx2-dependent gene, katanin-like 1 was investigated further by loss-of-function studies. Prior to this study the mtx2 gene was identified by homology, within its homeodomain, to other Mix-family transcription factors, but both its function and transcriptional targets remained unknown. Using a morpholino knockdown approach, this thesis demonstrates that Mtx2 is essential for vegetal movement (epiboly), but not specification, of the embryonic germ layers and extra-embryonic tissues during zebrafish gastrulation. The recruitment of filamentous actin (F-actin) to a punctate band at the blastoderm margin, was previously shown to be responsible for progression of epiboly. However, formation of this structure is demonstrated to be Mtx2-dependent. Microarray expression profiling of the Mtx2 loss-of-function phenotype was performed to screen for novel genes with roles in gastrulation. This approach identified Mtx2-dependent genes with roles in cytoskeletal dynamics, cell-cell adhesion and endocytosis and vesicular trafficking – processes known to be involved in morphogenetic movements. Many Mtx2-dependent genes are co-expressed with mtx2 in the extra-embryonic yolk syncytial layer (YSL), the teleost functional equivalent of mammalian visceral endoderm. The subset of Mtx2-dependent genes co-expressed with mtx2 and that contain Mtx2-binding sites within their 2kb proximal promoter represent the genes with the greatest likelihood of being direct Mtx2 transcriptional targets. A novel homologue of the microtubule severing protein Katanin, known as katanin-like 1 (katnal1) met all these conditions. Morpholino knockdown of Katnal1 demonstrates that like Mtx2, Katnal1 is essential for gastrulation in zebrafish. A cloned Katnal1mCherry fusion construct was observed to associate with microtubules, and demonstrated bi-directional trafficking around transfected mammalian cells. Analysis of the microtubule network in wild-type and morpholino injected zebrafish embryos demonstrated that remodelling of the extensive microtubule network found in the YSL and yolk cytoplasmic layer (YCL) is Katnal1-dependent. Nuclear division within the YSL and F-actin recruitment to the blastoderm margin are also Katnal1-dependent. This thesis therefore demonstrates, for the first time directly, the multiple, specific roles played by the microtubule network of the YSL/YCL. Katnal1 is highly conserved from Drosophila to mammals and is dynamically expressed during mouse gastrulation. The Mtx2 binding motif in the katnal1 2kb proximal promoter can be bound by both Mtx2 and its putative mouse homologue Mixl1. This suggests that katnal1 may also be a direct target of Mtx2. At the technical level, these results demonstrate the validity of screening for novel developmentally important genes using a zebrafish microarray-based approach, the potential of such an approach to, ab initio, identify a candidate list of transcription factor targets and confirm the utility of the zebrafish as a developmental model. At the biological level, this work collectively suggests that Mtx2 is a central regulator of the morphogenetic movement of epiboly and that Katnal1-dependent microtubule remodelling drives multiple aspects of gastrulation, potentially from Drosophila through to humans.

Le cytosquelette joue un rôle essentiel dans de nombreux processus cellulaires (division, adhésion, migration, morphogenèse..). Un de ses principaux constituants, les filaments d'actine, des polymères semi flexibles polarisés, forme des réseaux dont les architectures spécifiques permettent au cytosquelette de réaliser ses fonctions physiologiques. Un enjeu majeur en biologie cellulaire est de comprendre comment les cellules peuvent former une telle variété d'organisations à partir de la même entité de base, les monomères d'actine. Nous avons découvert récemment que limiter la nucléation des filaments d'actine à des géométries définies suffit à contrôler la formation de différentes organisations (Reymann et al, 2010). Néanmoins, les paramètres principaux permettant d'expliquer comment ces contraintes géométriques déterminent l'organisation collective des filaments n'ont pas été identifiés. Pour comprendre les lois physiques régissant ce phénomène, j'ai développé des simulations numériques du système expérimental en utilisant le logiciel Cytosim. J'ai pu ainsi montrer que la géométrie, les interactions stériques entre filaments, leurs propriétés mécaniques, et l'efficacité de la nucléation sont les paramètres clés contrôlant la formation de structures. Cette étude propose une base solide pour comprendre l'organisation cellulaire de l'actine en identifiant un système minimal de composants suffisant pour simuler l'émergence de différentes organisations d'actine (réseau branché, faisceaux de filaments parallèles ou antiparallèles). Avec cet outil, nous pouvons à présent prédire, étant donnée une géométrie de nucléation, quelles structures en émergeront.Nous avons alors combiné nos deux méthodes in-vitro et in-silico pour étudier comment le couplage entre l'architecture des réseaux et leur composition biochimique contrôle la réponse contractile. La connectivité entre les filaments en est un facteur crucial. En effet, un réseau peu connecté se déforme seulement localement, et n'instaure pas de comportement global. Une structure fortement connectée est très rigide, les moteurs moléculaires ne peuvent donc pas la déformer efficacement. La contraction d'une structure n'est donc possible que pour des valeurs de connectivité intermédiaires. L'amplitude de cette contraction est alors déterminée par l'organisation des filaments. Ainsi nous avons pu expliquer comment l'architecture mais aussi la connectivité des réseaux gouverne leur contractilité.Finalement, les microtubules sont aussi des acteurs essentiels aux processus cellulaires. Étant longs et rigides, ils servent de senseurs de la forme cellulaire et organisent les organites. Leur distribution spatiale, facteur majeur pour l'organisation cellulaire, est contrôlée dans un grand nombre de types cellulaires par la position du centrosome, un organite qui nuclée la plupart des microtubules. La capacité du centrosome à trouver le centre de la cellule dans de nombreuses conditions physiologiques est particulièrement étonante. Il peut aussi adopter une position décentrée lors de processus cellulaires spécifiques. Des mécanismes pouvant potentiellement expliquer le positionnement du centrosome ont été proposés (Manneville et al., 2006; Zhu et al, 2010), mais ce phénomène reste dans sa plus grande partie inexpliqué. J'ai utilisé les simulations pour explorer différents mécanismes pouvant le contrôler selon différentes conditions. Ces résultats permettent de disposer d'une base théorique pour présumer des mécanismes intervenant dans un système donné. Ils peuvent aussi permettre de valider ou réfuter des hypothèses sur les phénomènes mis en jeu et aider à l'élaboration de nouveaux systèmes expérimentaux.Les simulations que j'ai développées aident ici à étudier des comportements spécifiques, en apportant de nouveaux éclairages sur les comportements collectifs du cytosquelette. Elles pourraient être utilisées comme un outil prédictif ou adaptées pour l'étude d'autres systèmes expérimentaux. / The cytoskeleton plays a crucial role in cellular processes, including cell division, adhesion, migration and morphogenesis. One of its main compenent, the actin filaments, a polarised semi-flexible polymer, contributes to these processes by forming specific collective architectures, whose structural organisations are essential to perform their functions. A major challenge in cell biology is to understand how the cell can form such a variety of organisations by using the same basic entity, the actin monomers. Recently we discovered that limiting actin nucleation to specific regions was sufficient to obtain actin networks with different organization (Reymann et al., 2010). However, our understanding of the general parameters involved in geometrically-driven actin assembly was limited. To understand mechanistically how spatially constraining actin nucleation determines the emergent actin organization, I performed detailed simulations of the actin filament system using Cytosim, a simulation tool dedicated to cytoskeleton system. I found that geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. This study sets the foundation for our understanding of actin cellular organization by identifying a reduced set of components that were sufficient to realistically reproduce in silico the emergence of the different types of actin organization (branched actin network, parallel or anti parallel actin bundles). We can now predict for any given nucleation geometry which structures will form.Being able to control the formation of specific structures in-vitro and in-silico, we used the combination of both methods to study how the interplay between actin network architecture and its biochemical composition affects its contractile response. We highlighted the importance of the connectivity between filaments in the structures. Indeed, a loosely connected network cannot have a global behavior, but undergoes only local deformations. A highly connected network will be too rigid to be efficiently deformed by molecular motors. Only for an intermediate range of network connectivity the structures will contract, with an amplitude that depends notably on actin filaments organisation. This work explains how architecture and connectivity govern actin network contractility.Finally, the microtubules are also essential actors of cellular processes. Being long and rigid, they serve as sensors of the cellular shape and can organize the position of organelles in the cytoplasm. Their spatial distribution in the cell is thus a crucial cellular feature. this distribution is determined in a vast number of cell types by the position of the centrosome, an organelle that nucleates the majority of microtubules. Quite strinkingly, the centrosome is able to find the center of the cell in a lot of different physiological conditions, but can nonetheless adopt a decentered position in specific cellular processes. How this positioning is controled is not yet fully understood, but a few potential mechanims have been proposed (Manneville et al., 2006; Zhu et al., 2010). I used the simulations to explore different mechanisms taht can explain the position of the centrosome under different conditions. These results offer theorical considerations as a basis to assess which mechanism might prevail in a specific experimental system and may help to design new experimental setups.The simulations that I developed helped to study some specific behavior, by giving new insights into cytoskeleton collective organisations. These simulations can be further used as predictive tool or adapted to other experimental systems.

Les microtubules (MTs) sont importants pour des processus cellulaires majeurs comme la polarisation, le trafic membranaire, la division cellulaire ainsi que pour l'architecture intracellulaire. En retour, les organites influencent l'organisation et la dynamique des MTs. Mon projet de thèse vise à élucider les interactions fonctionnelles existantes entre les MTs et la sécrétion en adoptant une approche quantitative et systématique. En synchronisant le trafic de différents cargos grâce au système RUSH (Retention Using Selective Hooks) et à une dépolymérisation complète des MTs, nous avons montré que les MTs ne sont pas strictement nécessaires à la sécrétion des cargos. D'une manière générale nous avons observé que le trafic intracellulaire est ralenti mais toujours possible en présence d'un appareil de Golgi dispersé. Nous avons caractérisé deux populations d'éléments golgiens. Elles sont présentes peu après la dépolymérisation des MTs et sont différentes en terme de composition et de capacité de sécrétion. Nos résultats montrent qu'une maturation fonctionnelle des éléments golgiens est nécessaire pour le trafic post-golgien, basée sur l'acquisition de certains facteurs golgiens non identifiés à ce jour. Dans une deuxième partie, nous nous sommes intéressés à l'exocytose au niveau de la membrane plasmique. Nous avons observé une sécrétion préférentielle des protéines à proximité des adhésions focales. Différentes techniques de biologie cellulaire nous ont permis de caractériser cet adressage préférentiel vers les sites d'adhésion de la cellule. Nous avons également corrélé les forces exercées par la cellule sur le substrat avec la direction du transport antérograde. / Microtubules (MTs) are important for major cellular processes like cell polarization, intracellular trafficking, cell division, intracellular architecture. Organelles influence back the MTs organization and dynamics. The goal of my project was to study the involvement of MTs in the intracellular trafficking. Thanks to the Retention Using Selective Hooks (RUSH) system to synchronize the trafficking of cargos and with an efficient way to depolymerize MTs, we showed that MTs were not strictly essential to secretion of cargos. More generally, we showed that intracellular trafficking is slowed down but still possible in the presence of a dispersed Golgi apparatus. Moreover, we characterized two populations of Golgi elements in cells without MTs that are different in terms of secretion ability and composition. Our results demonstrated that functional maturation of Golgi elements is needed to ensure post-Golgi trafficking and that MTs driven post-Golgi transport is not strictly required. Besides working on intracellular trafficking without MTs, we conducted a study on the exocytosis at the plasma membrane. By using an antibody coating on coverslips to immobilize secreted cargos, we visualized the first step of arrival at the plasma membrane. We observed a directed and polarized secretion close to focal adhesions that we characterized by different cell biology technics and microscopy (spinning disk, TIRF…). We highlighted a close relationship between forces exerted by the cell on its substrate and the directionality of the anterograde transport by using patterning and Traction Force Microscopy (TFM).