Mitosis and meiosis are different forms of cell division. Mitosis is a non-reductive form of cell amplification whereby DNA chromosomes are replicated and segregated to form two progeny copies of the progenitor cell. Meiosis is a reductive form of cell division creating progeny containing half the chromosome copies of the progenitor cell. Improper chromosome segregation creates aneuploidy, which is poorly tolerated in cells. In cycling mitotic cells, aneuploidy leads to genome instability and cell death. Following meiosis, aneuploidy is associated with infertility, miscarriages, and birth defects. To segregate chromosome copies properly, pairs are physically organized and segregated to progeny cells by a mitotic spindle, whose functionality is tightly regulated. Kinesins are a family of highly conserved dimeric ATPase proteins which; organize spindle shape and size, facilitate chromosome capture and attachment to the spindle, and generate forces which are required for segregation. I investigated the molecular structure and function of human kinesin 13 family protein, Mitotic Centromere Associated Kinesin, MCAK. MCAK is a microtubule depolymerase whose full molecular structure and mechanism of depolymerization is not fully understood. Using in vitro biochemical assays and in vivo TIRF imaging, I found that altering MCAK molecular structure alters MCAK sub-spindle localization and by inference, alters global microtubule dynamics. This study suggests a potential mode for regulating of MCAK activity/function requiring further testing. Compared to over 30 kinesins in humans, showing a large amount of functional redundancy, yeast only has 6 identified kinesins whose function during meiotic cell division are still relatively unknown. I screened the importance and redundancy of yeast kinesins during meiosis. The results suggest similar roles and redundancies in meiosis to that during mitosis, despite different biochemical and biophysical spindle environments. Together, my investigations broaden the understanding of kinesin regulation and functional redundancy during different types of cell division.

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.

No description available.

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.

Les microtubules sont de longs polymères cylindriques de la protéine α, β tubuline, utilisés dans les cellules pour construire le cytosquelette, le fuseau mitotique et les axonèmes. Ces polymères creux sont cruciaux pour de nombreuses fonctions cellulaires, y compris le transport intracellulaire et la ségrégation chromosomique pendant la division cellulaire. Au fur et à mesure que les cellules se développent, se divisent et se différencient, les microtubules passent par un processus, appelé instabilité dynamique, ce qui signifie qu’ils basculent constamment entre les états de croissance et de rétrécissement. Cette caractéristique conservée et fondamentale des microtubules est étroitement régulée par des familles de protéines associées aux microtubules. Les protéines de kinésine-13 sont une famille de facteurs régulateurs de microtubules qui dépolymérisent catalytiquement les extrémités des microtubules. Cette thèse traite d’abord des concepts mécanistiques sur le cycle catalytique de la kinésine-13. Afin de mieux comprendre le mécanisme moléculaire par lequel les protéines de kinésine-13 induisent la dépolymérisation des microtubules, nous rapportons la structure cristalline d’un monomère de kinésine-13 catalytiquement actif (Kif2A) en complexe avec deux hétérodimères αβ-tubuline courbés dans un réseau tête-à-queue. Nous démontrons également l’importance du « cou » spécifique à la classe de kinésine-13 dans la dépolymérisation catalytique des microtubules. Ensuite, nous avons cherché à fournir la base moléculaire de l’hydrolyse tubuline-guanosine triphosphate (GTP) et son rôle dans la dynamique des microtubules. Dans le modèle que nous présentons ici, l’hydrolyse tubuline-GTP pourrait être déclenchée par les changements conformationnels induits par les protéines kinésine-13 ou par l’agent chimique stabilisant paclitaxel. Nous fournissons également des preuves biochimiques montrant que les changements conformationnels des dimères de tubuline précèdent le renouvellement de la tubuline-GTP, ce qui indique que ce processus est déclenché mécaniquement. Ensuite, nous avons identifié la kinésine de microtubule Kif2C comme une protéine associée à des modèles d’ADN imitant la rupture double brin (DSB) et à d’autres protéines de réparation DSB connues dans les extraits d’œufs de Xenope et les cellules de mammifères. Les cassures double brin d’ADN (DSB) sont un type majeur de lésions d’ADN ayant les effets les plus cytotoxiques. En raison de leurs graves impacts sur la survie cellulaire et la stabilité génomique, les DSB d’ADN sont liés à de nombreuses maladies humaines, y compris le cancer. Nous avons constaté que les activités PARP et ATM étaient toutes deux nécessaires pour le recrutement de Kif2C sur les sites de réparation de l’ADN. Kif2C knockout ou inhibition de son activité de dépolymérisation des microtubules a conduit à l’hypersensibilité des dommages à l’ADN et à une réduction de la réparation du DSB via la jonction terminale non homologue et la recombinaison homologue. Dans l’ensemble, notre modèle suggère que les protéines de kinésine-13 peuvent interagir avec les dimères de tubuline aux extrémités microtubules et modifier leurs conformations, moduler l’étendue des extrêmités tubuline-GTP dans les cellules et déclencher le désassemblage des microtubules. Ces deux modèles pourraient être des clés pour démêler les mécanismes impliqués dans le nouveau rôle de Kif2C dans la réparation de l’ADN DSB sans s’associer à des polymères de microtubules. / Microtubules are long, cylindrical polymers of the proteins α, β tubulin, used in cells to construct the cytoskeleton, the mitotic spindle and axonemes. These hollow polymers are crucial for many cellular functions including intracellular transport and chromosome segregation during cell division. As cells grow, divide, and differentiate, microtubules go through a process, called dynamic instability, which means they constantly switch between growth and shrinkage states. This conserved and fundamental feature of microtubules is tightly regulated by families of microtubule-associated proteins (MAPs). Kinesin-13 proteins are a family of microtubule regulatory factors that catalytically depolymerize microtubule ends. This thesis first discusses mechanistic insights into the catalytic cycle of kinesin-13. In order to better understand the molecular mechanism by which kinesin-13 proteins induce microtubule depolymerization, we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array. We also demonstrate the importance of the kinesin-13 class-specific “neck” in modulating Adenosine triphosphate (ATP) turnover and catalytic depolymerization of microtubules. Then, we aimed to provide the molecular basis for tubulin-Guanosine triphosphate (GTP) hydrolysis and its role in microtubule dynamics. Although it has been known for decades that tubulin-GTP turnover is linked to microtubule dynamics, its precise role in the process and how it is driven are now well understood. In the model we are presenting here, tubulin-GTP hydrolysis could be triggered via the conformational changes induced by kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We also provide biochemical evidence showing that conformational changes of tubulin dimers precedes the tubulin-GTP turnover, which indicates that this process is triggered mechanically. Next, we identified microtubule kinesin Kif2C as a protein associated with double strand break (DSB)-mimicking DNA templates and other known DSB repair proteins in Xenopus egg extracts and mammalian cells. DNA double strand breaks (DSBs) are a major type of DNA lesions with the most cytotoxic effects. Due to their sever impacts on cell survival and genomic stability, DNA DSBs are related to many human diseases including cancer. Here we found that PARP and ATM activities were both required for the recruitment of Kif2C to DNA repair sites. Kif2C knockdown/knockout or inhibition of its microtubule depolymerizing activity led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both non-homologous end-joining (NHEJ) and homologous recombination (HR). Interestingly, genetic depletion of KIF2C, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Altogether, our findings shed light on the mechanisms involved in kinesin-13 catalyzed microtubule depolymerization. Our tubulin-GTP hydrolysis model suggests that kinesin-13 proteins may interact with tubulin dimers at microtubules ends and alter their conformations, modulate the extent of the GTP caps in cells and trigger microtubule disassembly. These two models could be keys to unravel the mechanisms involved in the novel role of Kif2C in DNA DSB repair without associating with microtubule polymers.