Understanding how the cellular cytoskeleton is maintained and regulated is important to elucidate the functions of many structures such as the mitotic spindle, cilia and flagella. Katanin p60, microtubule-severing enzymes from the ATPase associated with cellular activities (AAA+) family, has previously been shown in our lab to be inhibited by free tubulin as well as α- and β-tubulin carboxy-terminal tail (CTT) constructs. Here we investigate the inhibition ability of several different tubulin CTT sequences. We quantify the effect of the addition of these constructs on the severing and binding activity of katanin. We find that some constructs inhibit katanin better than others and two constructs that appear to enhance katanin activity. Our findings add nuance to our previous findings that consensus α-tubulin tails are less inhibitory of katanin than consensus β-tubulin [3]. Surprisingly, we find that a polyglutamate sequence activates katanin while it has previously been shown to inhibit spastin, a different microtubule-severing enzyme associated with the neuromuscular disease Hereditary Spastic Paraplegia [23]. These results highlight that different CTT sequences can control the activity of severing enzymes and ultimately affect the cytoskeletal network organization in a cell type and location-dependent manner.

At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.

Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3. Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique. The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction). Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.

Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length.

Pericentrin and γ-tubulin are two resident centrosome proteins that are involved in microtubule nucleation and organization. When cytosolic extracts of Xenopus eggs were analyzed on sucrose gradients and gel filtration, the two proteins comigrated on gradients and co-eluted from the column. Immunodepletion of γ-tubulin removed all of the soluble pericentrin. The complex of the two proteins was estimated to be ~3-5 megaDaltons (MD), consisting of a pericentrin complex of ~20S and a γ-tubulin complex of ~25S, presumably the γ-TURC (~2 MD). When analyzed at the centrosome by enhanced deconvolution immunofluorescence the two proteins colocalized within a novel ring-like lattice structure, unlike other centrosome proteins analyzed, and were sufficiently close to generate FRET. The levels of the two proteins increased through the cell cycle, peaking at metaphase, and these changes were accompanied by structural changes in the lattice. Nucleated microtubules appeared to contact lattice elements throughout the centrosome. Inhibition of pericentrin function diminished assembly of γ-tubulin onto centrosomes, as did microtubule depolymerization and inhibition of dynein funciton. Separate fractions of the two proteins showed that pericentrin was required in the form a ~20S complex to bind γ-tubulin and for γ-tubulin assembly and microtubule nucleation. Overexpressed and purified pericentrin from cells eluted as a single polypeptide and was not competent to bind γ-tubulin. These results show that pericentrin in the context of a ~20S complex functions to assemble γ-tubulin into the centrosome lattice, and suggests that the pericentrin complex associated with the γ-TURC consists of an essential unit for centrosome formation.

The mechanism for centrosome assembly in somatic cells has previously been proposed to be microtubule independent. Studies presented in this dissertation demonstrate that in somatic cells pericentrin and γ tubulin, two paradigm centrosome proteins, assemble onto the centrosome in a microtubule, and dynein/dynactin dependent manner. High resolution, three-dimensional, time-lapse digital imaging of pericentrin-GFP labeled centrosomes has revealed tiny particles that move vectorally towards the centrosome at rates exceeding 1μm/second. These pericentrin-GFP particles contain γ tubulin and are not readily visible by standard two-dimensional digital imaging microscopy. Further studies have shown that dynein colocalizes with tiny particles of endogenous pericentrin outside of the centrosome which may reflect assembly intermediates in transit towards the centrosome. Furthermore, when dynein function is disrupted in G1 cells by nocodazole treatment, dynamitin overexpression, or dynein IC antibody (70.1) injection, assembly of pericentrin and γ tubulin onto the centrosome throughout the cell cycle is greatly reduced. Moreover, microtubule co-sedimentation studies have demonstrated that pericentrin associates with microtubules in vitro and is dependent on functional dynein/dynactin. Together these data strongly suggest that pericentrin and γ tubulin are novel cargoes of the dynein/dynactin motor complex which transports these proteins -and likely other components of the 3MDa nucleating complex (Dictenberg et al., 1998)- to the centrosome via rnicrotubules.

No description available.

Etude de l'implication de CRMP4, un partenaire de MAP6, dans la voie de signalisation sémaphorine 3E.Pendant le développement embryonnaire, les neurones établissent des milliards de connexions. Ces connexions ne sont pas aléatoires, mais précisément orientées, dirigées par des molécules de guidage situées dans l’environnement cellulaire. Le branchement inapproprié de ces neurones a de graves conséquences sur les fonctions sensorielles, motrices et cognitives du système nerveux, aboutissant à des pathologies neurologiques et psychiatriques telle que la schizophrénie. Ainsi la mutation de certaines protéines impliquées dans le guidage de ces connexions, comme MAP6 ou CRMP4, peut entraîner des perturbations conduisant à des prédispositions pour le développement de telles pathologies. En effet l'absence de MAP6 (souris KO MAP6) conduit à l'altération de nombreuse connections neuronales associé a différents troubles comportementaux réminiscent avec des symptômes schizoïdes. Parmi les faisceaux d'axones affectés on remarque la disparition du fornix, un faisceau neuronal connu pour son implication dans la schizophrénie. Cette disparition est en partie causée, en l'absence de MAP6, par l'abolition de la signalisation induite par la molécule de guidage sémaphorine 3E (Sema3E). Dans ce projet de thèse, le lien entre MAP6 et CRMP4 dans cette voie de signalisation Sema3E à pu être établi. De plus, l'impact de l'absence de la protéine CRMP4 sur la formation du fornix a pu être caractérisé par l'étude neuroanatomique des souris KO CRMP4. Nous avons par ailleurs pu mettre en évidence de nouvelles altérations causée par l'absence de MAP6. Dans son ensemble ce travail approfondit les connaissances des défauts des connectivités des souris KO MAP6 et identifie CRMP4 comme un nouvel acteur de la signalisation Sema3E et de la formation du fornix. / Study of the involvement of the MAP6 partners, CRMP4, in the semaphorin 3E signaling pathway.During embryonic development, neurons establish billion of connections between them. Those connections are not random. On the contrary, they are precisely targeted thanks to the driving by cellular environment guidance cues. A wrong branching of those neurons can lead to dramatic impairment of sensory, motor and cognitive function of the central nervous system resulting in neurologic or psychiatric disorders such as Schizophrenia. Thus, mutation of proteins implicated on neurons guidance like MAP6 or CRMP4 can lead to susceptibility for those kind of pathology occurrence. In fact, MAP6 deletion ( MAP6 KO mice) leads to diverse neuronal connectivity alterations associated to schizophrenia-like behavior disorders. Among axonal tracts affected we notice the absence of the fornix known for its implication on Schizophrenia. In MAP6 KO mice, this fornix disruption is partly due to the loss of semaphorin 3E (Sema3E) dependant signaling pathway. This project shows the involvement of CRMP4, a partner of MAP6, in the Sema3E signaling pathway. Furthermore, it characterized the impact of the CRMP4 deletion (CRMP4 KO) on fornix formation. Finally, neuroanatomical studies allowed us to identify unknown alteration of MAP6 KO mice connectivity alteration.

Les agents ciblant les microtubules sont des médicaments anticancéreux efficaces. Leur utilisation dans le cadre d’un traitement combiné avec des rayonnements ionisants est également une stratégie prometteuse. Cependant, l’apparition de résistances aux produits chimiques et aux radiations nécessite de rechercher d'autres types de traitements. Nos laboratoires ont récemment décrit deux médicaments qui ciblent directement ou indirectement les microtubules. Premièrement, un analogue du 2-méthoxyestradiol, un poison de fuseau se liant à des microtubules et provoquant la formation de fuseaux mitotiques anormaux. Il s'agit du 2-éthyl-3-O-sulphamoyle-estra-1,3,5 (10) 16-tétraène (ESE-16). Deuxièmement, le 9-benzoyloxy-5,11-diméthyl-2H, 6H-pyrido [4,3-b] carbazol-1-one (LimPyr1), un nouvel inhibiteur des LIM kinases induisant indirectement la stabilisation des microtubules. Il a été démontré récemment que LimPyr1 est actif sur les modèles de cancer du sein résistants au taxol. En tant que médicaments ciblant les microtubules, les deux agents, ESE-16 et LimPyr1, induisent des défauts mitotiques. Nous émettons donc l’hypothèse qu’ils pourraient sensibiliser les cellules aux radiations. Le but de ce projet de thèse était de vérifier cette hypothèse et, plus précisément, de déterminer si de faibles doses de ESE-16 et de LimPyr1 pourraient augmenter l'apoptose et retarder la réparation nucléaire induite par le rayonnement dans les cellules du cancer du sein in vitro.Différentes lignées cellulaires cancéreuses, les cellules MCF-7, MDA-MB-231 et BT-20, ont été exposées à ESE-16 et à LimPyr1 pendant 24 heures avant un rayonnement de 8 Gy. Les effets de ces combinaisons thérapeutiques ont été comparés à ceux obtenus à partir de cellules exposées aux composés seuls ou aux seules radiations. L'activation des voies de survie et des voies apoptotiques intrinsèques a été étudiée. Les résultats ont révélé une augmentation de la signalisation de la survie et de la mort dans les cellules exposées aux traitements individuels. Les traitements combinés ont diminué la survie des cellules alors que la signalisation apoptotique augmentait, entraînant une augmentation de l'apoptose. En outre, les traitements combinés ont augmenté de manière significative la présence de micronoyaux dans les cellules BT-20, indiquant une augmentation des dommages à l'ADN. Les cellules MCF-7 et MDA-MB-231 présentent une formation de micronoyaux similaire lorsqu'elles sont exposées à la combinaison de traitements ou au rayonnement uniquement. La phosphorylation de H2AX (γH2AX) (normalement augmentée lors de dommages à l'ADN) et l'expression de Ku70 (nécessaire pour la réparation de l'ADN) étaient diminuées dans les cellules de cancer du sein prétraitées 2 heures après l'irradiation par rapport aux cellules exposées à l'irradiation uniquement. L'expression de H2AX et Ku70 est cependant significativement accrue 24 heures après irradiation des cellules prétraitées par rapport aux cellules exposées aux traitements individuels. Des expériences portant sur la réponse adaptative ont révélé que LimPyr1 diminuait le développement de la résistance aux radiations en augmentant la perméabilité transmembranaire mitochondriale et en générant des ROS, un mécanisme qui n'est pas observé dans cellules traitées par ESE-16. Nous avons également observé une communication intercellulaire entre les cellules exposées au rayonnement et les cellules non exposées via l'effet induit par le rayonnement.En conclusion, le blocage mitotique partiel induit par ESE-16 et LimPyr1 rend les chromosomes plus exposés aux dommages dus aux radiations, comme l'indique l'augmentation de la présence de micronoyaux. De plus, les deux composés diminuent la signalisation et le trafic des protéines de protection et de dommages à l'ADN. En outre, LimPyr1 empêche le développement de résistances aux radiations dans les cellules exposées aux radiations. / Microtubule targeting agents are effective anti-cancer drugs. Their use as part of a combined treatment modality with ionising radiation is also a promising strategy. However, the emergence of resistance to chemical and radiation requires searching for alternative treatments. Our laboratories have recently described two drugs that directly or indirectly target the microtubules. Firstly, an analogue of 2-methoxyestradiol, a spindle poison binding to microtubules and causing the formation of abnormal mitotic spindles. This is 2-ethyl-3-O-sulphamoyl-estra-1,3,5 (10) 16-tetraene (ESE-16). Secondly, 9-benzoyloxy-5,11-dimethyl-2H, 6H-pyrido [4,3-b] carbazol-1-one (LimPyr1), a novel inhibitor of LIM kinases indirectly inducing microtubule stabilization. It has been recently shown that LimPyr1 is active on taxol-resistant breast cancer models. As microtubule-targeting drugs, both agents, ESE-16 and LimPyr1, induce mitotic defects. We thus hypothesize that they could sensitize cells to radiation. The aim of this PhD project was to test that hypothesis and, more specifically, to investigate whether low-dose ESE-16 and LimPyr1 could increase apoptosis and delay nuclear repair induced by radiation in breast cancer cells in vitro.Various cancer cell lines, MCF-7-, MDA-MB-231- and BT-20 cells, were exposed to ESE-16 and LimPyr1 for 24-hours prior to 8 Gy radiation. The effects of these combination therapies were compared to those obtained from cells exposed to the compounds alone or only to radiation. The activation of the survival and intrinsic apoptotic pathways were investigated. Results revealed an increase in survival and -death signaling in cells exposed to the individual treatments. The combination treatments decreased the cell survival while apoptotic signaling was increased, resulting in increased apoptosis. Furthermore, the combination treatments significantly increased the presence of micronuclei in BT-20 cells, indicating an increase in DNA damage. MCF-7- and MDA-MB-231 cells displayed similar micronuclei formation when exposed to the combination treatments or radiation only. Phosphorylation of H2AX (γH2AX) (normally increased upon DNA damage) and Ku70 expression (required for DNA repair) were decreased in pretreated breast cancer cells 2 hours after irradiation compared to cells exposed to irradiation only. The expression of H2AX and Ku70, however, is significantly increased 24 hours after irradiation of the pretreated cells relative to the cells exposed to the individual treatmentsExperiments investigating the adaptive response revealed that LimPyr1 decreased radiation resistance development by increasing the permeability of the mitochondrial transmembrane (flow cytometry measuring Mitocapture™) and the generation of ROS (flow cytometry employing hydroethidine), a mechanism not observed in ESE-16 pre-treated cells. We also observed an intercellular communication between cells exposed to radiation and non-exposed cells via the radiation induced bystander effect.In conclusion, the anti-mitotic effect of ESE-16 and LimPyr1 renders the chromosomes more exposed to radiation damage, as assessed by the increased occurrence of micronuclei. Moreover, both compounds decrease the signaling and trafficking of DNA damage and repair proteins. Additionally, LimPyr1 prevented the development of radiation resistance in cells exposed to radiation.

No description available.