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Schaedel, Laura. "Les propriétés mécaniques des microtubules". Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAY010/document.
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Les microtubules-qui définissent la forme des axones, des cils et des flagelles, et qui servent de rails pour le transport intracellulaire-subissent de fortes contraintes exercées par les forces intracellulaires. La structure des microtubules et leur rigiditépeuvent en théorie être affectées par des contraintes physiques. Cependant, il reste à établir comment les microtubules tolèrent de telles forces et quelles sont les conséquences de ces forces sur la structure des microtubules. En utilisant un dispositif demicrofluidique, j’ai pu montrer que la rigidité des microtubules diminue progressivementà chaque cycle de courbure induit par des contraintes hydrodynamiques.Comme dans d'autres exemples de fatigue des matériaux, l'application de contraintes mécaniques sur des défauts pré-existants le long des microtubules est responsable de la génération de dommages plus étendus. Ce processus rend les microtubules moins rigides.J’ai pu aussi montrer que les microtubules endommagés peuvent se réparer en intégrant de nouveaux dimères de tubuline à leur surface et de récupérer ainsi leur rigidité initiale. Nos résultats démontrent que les microtubules sont des matériaux biologiquesayant des propriétés d’auto-réparation, et que la dynamique des microtubules ne se produit pas exclusivement à leurs extrémités. La mise en évidence de ces nouvelles propriétés permet de montrer comment les microtubules peuvent s’adapter à des contraintesmécaniquesMicrotubules—which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport—can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules’ adaptation to mechanical stresses
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A, S. Jijumon. "Systematic characterization of a large number of Microtubule-Associated Proteins using purification-free TIRF-reconstitution assays Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles Microtubule-Associated Proteins: Structuring the Cytoskeleton Purification of custom modified tubulin from cell lines and mouse brains by polymerization-depolymerization cycles". Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL007.
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Le cytosquelette des microtubules (MTs) est constitué de filaments dynamiques impliqués dans une multitude de fonctions telles que la division cellulaire, le maintien de forme des cellules, les battements ciliaires ou encore la différenciation neuronale. Une régulation stricte des fonctions des MTs est donc d'une grande importance pour l'homéostasie cellulaire, et toute perturbation pourrait potentiellement conduire à des maladies comme le cancer, les ciliopathies ou la neurodégénérescence. Dans un contexte cellulaire, les propriétés des MTs peuvent être contrôlées par leurs interactions avec une grande variété de protéines associées (MT-associated proteins ; MAPs). Notre connaissance de ces interacteurs s'est continuellement enrichie au cours des dernières décennies, mais il n'existe à ce jour aucune étude systématique visant à décrire et à classer ces protéines en fonction de leurs mécanismes de liaison et de leurs effets structuraux sur les MTs. Dans mon projet de thèse, j’ai mis au point un essai permettant une analyse rapide et systématique à la base des lysats clarifiés de cellules humaines surexprimant une multitude des différents MAPs. Le comportement dynamique des MT en présence d'environ 50 MAPs différentes a été imagé à l'aide de la microscopie TIRF. Cela nous permet d'étudier le comportement des MAP dans une situation proche de leur environnement naturel, mais en éliminant la complexité de l'espace intracellulaire, telle que l'encombrement par des organelles et des filaments du cytosquelette à l'intérieur de l'espace intracellulaire confiné. En effet, la plupart des MAPs étaient bien solubles dans notre approche d'extraction, tandis que les approches de purification pour plusieurs d'entre elles ont conduit à leur précipitation, rendent les expériences de reconstitution in vitro classique impossible. Ma nouvelle approche m’a permis de définir plusieurs nouvelles protéines comme de véritables MAP. J’ai montré que des MAPs non-caractérisées auparavant ont des effets étonnamment différents sur la polymérisation et la structure des MTs, créant ainsi une variété de réseaux de MT distincts. J’ai également démontré que mon approche permet d'étudier les structures des MAPs associées aux MTs par cryo-microscopie électronique, ou d'étudier le dynamique des MTs porteuses de mutations trouvées dans les pathologies humaines. J’ai également démontré que mon approche permet à tester la sensibilité des MAPs aux modifications post-traductionnelles de la tubuline, ou d'étudier le rôle des MAPs dans les interactions entre l'actine et les MTs. Mon approche expérimentale permet donc de mieux comprendre comment les MAP et les MT contrôlent ensemble le fonctionnement du cytosqueletteMicrotubules (MTs) are dynamic filaments involved in a plethora of functions such as cell division, cell shape, ciliary beating, neuronal differentiation. Strict regulation of MT functions is therefore of high importance for the cellular homeostasis, and any perturbations could potentially lead to diseases like cancer, ciliopathies and neurodegeneration. At the protein level, there are accumulating studies showing that MT properties can be controlled via interaction with a large variety of MT-associated proteins (MAPs). Our knowledge of MAPs has been enriched over time, but up to this date no systematic studies exist that aim to describe and categorize these proteins according to their binding mechanisms and structural effects on MTs. In my PhD project, I have developed an assay for rapid and systematic analysis of MAPs using cleared lysates of cultured human cells in which I overexpress a variety of different MAPs. The dynamic behaviour of growing MTs in the presence of those MAPs were imaged using TIRF microscopy. This allows me to study the behaviour of around 50 MAP candidates in a situation close to their natural environment, but eliminating complexity coming from different organelles and crammed cytoskeleton filaments inside the confined intracellular space. Indeed, most MAPs were nicely soluble in the extract approach, while purification attempts of several of them led to protein precipitation, thus making classical invitro reconstitution approaches impossible. This novel approach allowed me to compare many MAPs under similar experimental conditions, and helped to define several novel proteins as bona-fide MAPs. I demonstrate that previously uncharacterized MAPs have strikingly different effects on MT polymerization and MT structure, thus creating a variety of distinct MT arrays. I further extended this cell-free pipeline to study structures of MAPs bound to MTs by cryo-electron microscopy, or to study the MT interactions of MAPs carrying patient mutations. Finally, I demonstrated that my approach can be used to test the sensitivity of MAPs to tubulin PTMs, as well as to study the role of MAPs in actin-MT crosstalk. In the future, this novel approach will allow for a better mechanistic understanding of how MAPs and MTs together control cytoskeleton functions
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Jiang, Nan. "Exploring Microtubule Structural Mechanics through Molecular Dynamics Simulations". University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504878667194719.
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Melbinger, Anna Tatjana. "On the role of fluctuations in evolutionary dynamics and transport on microtubules". Diss., Ludwig-Maximilians-Universität München, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-148246.
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Swoger, Maxx Ryan. "Computational Investigation of Material and Dynamic Properties of Microtubules". University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1532108320185937.
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Paulin-Levasseur, Micheline. "Cellular dynamics of vimentin filaments and their spatial relationship to microtubules in lymphocytes". Thesis, University of Ottawa (Canada), 1987. http://hdl.handle.net/10393/5396.
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Sousa, Da Costa Maria Judite. "Csi2 modulates microtubule dynamics and helps organize the bipolar spindle for proper chromosome segregation in fission yeast". Paris 6, 2013. http://www.theses.fr/2013PA066626.
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Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms such as the spindle assembly checkpoint and centromere positioning further help to ensure complete segregation fidelity. We present here the fission yeast csi2+. Csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. The bipolar mitotic spindle contains many short dynamic microtubules of ~1 micron scale, this represents a challenge for live cell imaging because the typical maximum resolution of the optical microscope is ~λ/2 or ~300 nm. We developed a novel method to image short fission yeast mitotic microtubules using the thermosensitive reversible kinesin-5 cut7. 24ts to create monopolar spindles. Csi2-deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and a subsequent high rate of chromosome segregation defects. As csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. Our data suggests that the major determinant of chromosome segregation defects may be microtubule dynamic defectsLa ségrégation correcte des chromosomes est processus fondamental pour maintenir la stabilité génomique. Des défauts de ségrégation sont souvent à l’origine de l’apparition de cellules aneuploïdes, caractéristique fréquemment observée dans les cellules cancéreuses. Dans les cellules eucaryotes, la ségrégation correcte des chromosomes est assurée par le fuseau mitotique. Des mécanismes de contrôle, tels que le point de contrôle mitotique et le bon attachement des centromères, sont mis en œuvre pour assurer la bonne ségrégation des chromosomes. Dans cette étude, nous avons pu établir chez le levure fissipare, que la protéine csi2, localisée aux pôles du fuseau mitotique, joue un rôle sur la dynamique des MTs mitotiques, dans la formation d’un fuseau mitotique intègre et par conséquent dans la ségrégation correcte des chromosomes. Les MTs composants le fuseau mitotique bipolaire sont dynamiques et de petite taille ~1µm ce qui représente un défis technique pour les imager, en effet, la résolution optique d’un microscope ~λ/2 est en général de 300nm. Nous avons développé une nouvelle approche pour imager les MTs mitotiques basée sur l’utilisation du mutant réversible thermosensible kinesin-5 cut7. 24ts, pour obtenir des cellules ayant des fuseaux monopolaires. Ainsi, nous avons pu mettre en évidence que la délétion de la protéine csi2 chez la levure S. Pombe était à l’origine d’un allongement de la longueur des microtubules mitotiques, d’une augmentation du nombre de cellules présentant un fuseau monopolaire et d’une augmentation des défauts de ségrégation des chromosomes. L’étude de l’implication de la protéine csi2 dans ces différents mécanismes nous a permis de mettre en évidence la contribution de chacun de ces mécanismes dans la bonne ségrégation des chromosomes. Nous proposons dans cette étude que le facteur déterminant à l’origine d’une ségrégation incorrecte des chromosomes serait majoritairement imputable à des défauts de régulation de la dynamique des microtubules
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Ng, Daniel. "Investigating the dynamics of adhesion complex turnover by mass spectrometry based proteomics". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-dynamics-of-adhesion-complex-turnover-by-mass-spectrometry-based-proteomics(4e6d3051-c007-4715-a290-9acfd45d38a7).html.
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Adhesion complexes (ACs) are large macromolecular complexes of integrins and associated proteins that connect the actin cytoskeleton to the extracellular matrix. In migrating cells, ACs are highly dynamic -- forming and maturing at the cell front and disassembling at the cell rear. The turnover of ACs enables and localises the necessary traction forces required for cell migration. There is evidence for the spatiotemporal recruitment of specific proteins during AC maturation or disassembly; however, a holistic understanding of the compositional changes to ACs during these states is lacking. To this end, we sought to characterise the dynamic changes that occur at ACs during turnover using a mass spectrometry (MS)-based proteomics approach. A major challenge in studying AC turnover is the desynchronised nature of AC formation, maturation and disassembly within a population of cells. Therefore a nocodazole-washout assay was used to synchronise microtubule-induced AC maturation and disassembly. To study the dynamics of AC turnover by MS, an AC isolation method was optimised for use with the nocodazole-washout assay. Subsequently, the maturation of ACs by the loss of microtubules was studied by MS-based proteomics, and it was found that this resulted in the overall accumulation of adhesion proteins, and also the conversion of fibrillar adhesions to focal adhesions. Studying the dynamic process of AC disassembly requires a sensitive MS quantification method; as such, label-free quantitative methods were compared, and it was found that LC-MS peak ion intensity quantification performed better than spectral counting. Using optimised methodologies for isolation of ACs and MS quantification, the dynamics of AC disassembly was analysed over the course of the nocodazole-washout assay. It was found that in general, microtubules were enriched around ACs, whereas many structural AC proteins decreased over time. In summary, we have optimised methods for the study of ACs by MS-based proteomics, and applied these methods to the study of AC turnover.
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Rauch, Philipp. "Neuronal Growth Cone Dynamics". Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-119885.
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Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement.
When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity.
Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.
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Shukla, Nandini Y. "Investigation of Microtubule dynamics and novel Microtubule-associated proteins in growth and development of the filamentous fungus, Aspergillus nidulans". The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu149276142029341.
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Ebbinghaus, Maximilian. "Stochastic modeling of intracellular processes : bidirectional transport and microtubule dynamics". Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00592078.
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This thesis uses methods and models from non-equilibrium statistical physics to describe intracellular processes. Bidirectional microtubule-based transport within axons is modeled as a quasi-one-dimensional stochastic lattice gas with two particle species moving in opposite directions under mutual exclusion interaction. Generically occurring clusters of particles in current models for intracellular transport can be dissolved by additionally considering the dynamics of the transport lattice, i.e., the microtubule. An idealized model for the lattice dynamics is used to create a phase transition toward a homogenous state with efficient transport in both directions. In the thermodynamic limit, a steady state property of the dynamic lattice limits the maximal size of clusters. Lane formation mechanisms which are due to specific particle-particle interactions turn out to be very sensitive to the model assumptions. Furthermore, even if some particle-particle interaction is considered, taking the lattice dynamics into account almost always improves transport. Thus the lattice dynamics seems to be the key aspect in understanding how nature regulates intracellular traffic. The last part introduces a model for the dynamics of a microtubule which is limited in its growth by the cell boundary. The action of a rescue-enhancing protein which is added to the growing tip of a microtubule and then slowly dissociates leads to interesting aging effects which should be experimentally observable.
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Sears, James Cooper. "FoxO Regulates Microtubule Dynamics and Polarity to Promote Dendrite Branching in Drosophila Sensory Neurons". Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1476705366788041.
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Nolte, Elsie. "Etude du potentiel pro-apoptotique et radiosensibilisateur de quatre candidats-médicaments régulateurs des microtubules, sur des cellules de cancer du sein". Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV002.
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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 radiationsMicrotubule 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
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Johann, Denis [Verfasser] y Karsten [Akademischer Betreuer] Kruse. "Collective dynamics of molecular motors and passive cross-linkers on microtubules / Denis Johann. Betreuer: Karsten Kruse". Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2016. http://d-nb.info/110417037X/34.
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Abou, Serhal Daou Pascale. "The role of the diaphanous-related formins DRF1, DRF2 and DRF3 in ErbB2-dependent cell motility and microtubule dynamics". Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM5037.
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Les formines de la famille des DRF sont des puissants nucleateurs d'actine. Précédemment, nous avons montré que DRF1 participe à la capture des microtubules (MTs) au niveau du cortex cellulaire, en aval du récepteur ErbB2. Ceci impliquait le recrutement d'APC et ACF7. Dans cette étude, nous avons examiné la contribution de DRF1, DRF2 et DRF3 à la capture des MT corticaux et à la migration cellulaire ErbB2- dépendante. La déplétion individuelle de DRF1/2 ou 3 à l'aide de siRNA perturbe fortement la migration chimiotactique ErbB2-dépendante. Les DRF sont toutes trois requises pour la capture des MT au niveau du cortex cellulaire. Des mutants de DRF1 déficients pour leur association avec l'actine sont toujours actifs pour la capture des MT. Nous avons aussi pu montrer qu'une construction limitée au domaine FH2 des DRF était parfaitement fonctionnelle. Nous avons alors procéder à une recherche systématique des protéines se liant au domaine FH2, par purification d'affinité et spectrométrie de masse. Nous avons observé que les domaines FH2 de DRF1, DRF2 et DRF3 se lient à des groupes de partenaires distincts. Ainsi, seul le domaine FH2 de DRF1 lie la protéine Rab6-Interacting Protein 2 (RB6IP2). De plus, DRF1 contrôle le recrutement de RB6IP2 au cortex cellulaire et la déplétion concomitante de RB6IP2 et d'IQGAP1 perturbe fortement la capture des MT. Ces résultats démontrent l'implication de l'interaction entre DRF1 et RB6IP2 dans la capture des MT dans les cellules en migrationDiaphanous-related formins (DRF) nucleate single linear filaments, binding to and protecting from capping their growing barbed ends. We have previously found that DRF1 participated to the tethering of microtubules (MTs) to the cell cortex, downstream of the ErbB2 receptor tyrosine kinase. This involved the recruitment of APC and ACF7. We have now further investigated the contribution of DRF1, and of the closely related DRF2 and DRF3, to the capture of cortical MTs and ErbB2-dependent breast carcinoma cell migration.Using siRNA to knock down individual DRFs, we found that depletion of DRF1/2 or3 strongly disturbed ErbB2-dependent chemotaxis. All three DRFs were required for the formation of cortical MTs, in a non-redundant manner. DRF1 mutant proteins defective for actin binding were still active for MT capture. We also found that, upon truncation of the Formin Homology (FH) 1 domain, the FH2 domain remained fully functional. In a systematic search for proteins binding to the FH2 domains via affinity purification and mass spectrometry analysis, we observed that the FH2 domains of DRF1, DRF2 and DRF3 engaged with distinct sets of proteins. For instance, only FH2 domain of DRF1 pulled down Rab6-Interacting Protein 2 (RB6IP2). Interestingly, DRF1 controlled the cortical localization of RB6IP2 and concomitant depletion of RB6IP2 and IQGAP1 strongly disturbed capture of cortical MTs, showing the involvement of the DRF1/IQGAP1/RB6IP2 interaction in MT tethering at the cell leading edge
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Zhao, Bing [Verfasser] y Wolfram [Akademischer Betreuer] Brune. "The Role of Centrosomal Microtubules in F-actin Dynamics during Neuronal Polarization / Bing Zhao ; Betreuer: Wolfram Brune". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://d-nb.info/1182537804/34.
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Hirst, William Graham. "Tubulin biochemistry confers intrinsic differences in microtubule dynamics and drug sensitivity between species". Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22956.
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Mikrotubuli sind filamentöse intrazelluläre Polymere, die als grundlegende Bestandteile subzellulärer Strukturen in Eukaryoten dienen. Diese Studie verwendet einen vergleichenden Ansatz, um zu untersuchen, wie sich die intrinsischen dynamischen und biochemischen Eigenschaften von Tubulin zwischen verschiedenen Spezies unterscheiden, und zeigt ihre Konsequenzen in zwei verschiedenen physiologischen Kontexten: 1) Bestimmung der Spindelgröße bei Fröschen der Gattung Xenopus und 2) Spezifität von Mikrotubuli-Inhibitoren für Plasmodium falciparum-Mikrotubuli über denen ihres menschlichen Wirts.
In den Eiern der Froschgattung Xenopus wird die Länge der meiotischen Spindel biochemisch festgelegt und erreicht unabhängig von räumlichen Einschränkungen eine Obergrenze. Messungen der Dynamik von Xenopus-Mikrotubuli zeigen, dass X. laevis-Mikrotubuli sowohl schneller wachsen als auch länger leben als die von X. tropicalis. Darüber hinaus spielt die Quantifizierung der Länge und Massenverteilung der Xenopus-Mikrotubuli zusammen mit den Reaktionen der Eiextrakt-Spindelanordnung eine Rolle für die intrinsische Dynamik der Mikrotubuli bei der Modulation der Spindellänge.
Mikrotubuli sind auch Wirkstofftargets bei Pilz- und parasitären Helmintheninfektionen und haben in den letzten Jahrzehnten die Aufmerksamkeit als potenzielles Wirkstoffziel beim Malariaparasiten Plasmodium falciparum auf sich gezogen. Um die Dynamik und Medikamentspezifität von Mikrotubuli von P. falciparum zu charakterisieren, haben wir Tubulin direkt von den Parasiten gereinigt. Zum ersten Mal wurden hier dynamische P. falciparum-Mikrotubuli in vitro rekonstituiert und eine parasitenspezifische Unterdrückung der Dynamik von Mikrotubuli durch Oryzalin und Amiprofos-Methyl direkt nachgewiesen. Diese Studie legt einen experimentellen Rahmen fest, um direkt auf parasitenspezifische Hemmung von Mikrotubuli zu testen, die bisher unter Verwendung bestehender in-vitro-Ansätze nicht beobachtet wurden.Microtubules are filamentous intracellular polymers that are fundamental components of subcellular structures including the spindle, the cytoskeleton, and flagella in eukaryotes. This study uses a comparative approach to investigate how the intrinsic dynamic and biochemical characteristics of tubulin vary between species and demonstrates their consequences in two different physiological contexts: 1) Spindle size control in Xenopus frogs, and 2) The specificity of microtubule inhibitors for Plasmodium falciparum microtubules over those of their human host. In Xenopus frog eggs, the length of the spindle is biochemically controlled and reaches an upper limit independent of spatial constraints. In this study, in vitro measurements of Xenopus microtubule dynamics show that X. laevis microtubules are both faster-growing and longer-lived X. tropicalis, independent of the influence of microtubule-associated proteins. Furthermore, quantification of Xenopus microtubule length and mass distributions, combined with egg extract spindle assembly reactions, establishes a role for intrinsic microtubule dynamics in modulating spindle length. Microtubules are also established drug targets in fungal and parasitic helminth infections and have in the past decades drawn attention as a potential drug target in the malaria parasite Plasmodium falciparum. In order to characterize P. falciparum microtubule dynamics, structure, and drug specificity, we have used an affinity chromatography-based approach to purify tubulin directly from blood-stage parasites. For the first time, dynamic P. falciparum microtubules have been reconstituted in vitro and parasite-specific suppression of microtubule dynamics by oryzalin and amiprofos methyl has been directly demonstrated. This study establishes an experimental framework to directly test for parasite-specific microtubule inhibition, microtubule structure, and interactions with MAPs that previously have not observed using existing in vitro approaches.
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Dupont, Marie. "Étude fonctionnelle de gènes candidats impliqués dans les ciliopathies Human IFT52 mutations uncover a novel role for the protein in microtubule dynamics and centrosome cohesion". Thesis, Sorbonne Paris Cité, 2019. http://www.theses.fr/2019USPCB006.
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Les ciliopathies sont des maladies multisystémiques dues à des mutations dans des gènes codant des protéines localisées au cil. Les cils sont des organites constitués de microtubules présents à la surface de quasi toutes les cellules de vertébrés. IFT52 est un composant majeur du complexe du transport intraflagellaire IFT-B, indispensable à la formation et fonction du cil primaire. Des mutations faux-sens, non-sens ou de délétion en phase ont été identifiées dans IFT52 chez des individus présentant des phénotypes distincts de ciliopathies : une dysplasie rénale multikystique (DRK) ou des dysplasies squelettiques de type côtes courtes-polydactylie (CCP) ou Sensenbrenner (SB). Afin de mieux comprendre la variabilité phénotypique, nous avons étudié la pathogénicité de chacune des mutations grâce à des modèles cellulaires et animaux. D'abord, j'ai montré que les mutations de délétion en phase (CCP) et non-sens (SB), mènent en fait respectivement à un décalage de phase de lecture et un saut d'exon en phase, ce qui permettait d'expliquer en partie les relations génotype/phénotype. Par ailleurs, l'analyse des mutations faux-sens CCP et DRK montrait que la mutation CCP avait un effet plus délétère sur la fonction de IFT52 que celle de DRK, confirmant leur pathogénicité et expliquant le phénotype squelettique du cas CCP. Toutefois, cela n'expliquait pas la variabilité du phénotype rénal observée parmi les individus. De fait, nous avons considéré une deuxième mutation homozygote faux-sens présente dans le cas DRK dans le gène UBE2C, codant une enzyme E2 de conjugaison d'ubiquitine impliquée dans la sortie de mitose. La modélisation de la structure 3D de la protéine a montré que la mutation affectait la liaison à l'ubiquitine et de fait, probablement la fonction de la protéine. Des études in vitro ont confirmé que la mutation ralentissait la sortie de mitose des cellules. Par ailleurs, afin d'étudier l'impact de la mutation sur le développement rénal, une souris portant la mutation DRK (knock-in) a été générée par CRISPR/Cas9. Bien que les souris Ube2cKI/KI présentent un défaut de croissance et une létalité précoce similaire au modèle knock-out, validant ainsi la pathogénicité de la mutation, les reins sont normaux chez ces animaux. Nous avons donc émis l'hypothèse que le phénotype rénal résultait de la synergie entre les mutations IFT52 et UBE2C. Afin de tester cette hypothèse, des études de synergie ont été réalisées chez le poisson zèbre, par injection de morpholino ift52 dans des embryons issus du croisement d'animaux ube2c+/-. Cette approche n'a malheureusement pas permis d'induire un défaut rénal chez les embryons ube2c-/-. Ainsi soit le cas DRK présente une autre mutation pouvant expliquer le phénotype rénal, soit les modèles animaux (souris, poisson zèbre) ne sont pas appropriés à l'étude de l'implication de UBE2C dans le développement rénal humain. De manière inattendue, l'étude de IFT52 a permis de mettre en évidence un espacement anormal des centrioles composant le centrosome des cellules Ift52-/-. La cohésion du centrosome est maintenue par deux mécanismes : le lien protéique entre les parties proximales des centrioles, et la force des microtubules (MTs) qui s'exerce sur le centrosome. Aucune altération des principaux composants du lien protéique, notamment c-Nap1 et rootletine, n'a pu être observée dans les cellules Ift52-/-. En revanche, nous avons montré que Ift52 interagissait et co-localisait partiellement avec centrine au niveau de la partie distale du centrosome, suggérant un rôle de IFT52 dans le mécanisme des MTs. En effet, nous avons montré que les MTs présentaient un défaut d'ancrage au centrosome et qu'ils étaient moins dynamiques dans les cellules Ift52-/-. Nos résultats démontrent un rôle extra-ciliaire de IFT52 dans l'ancrage des MTs et la cohésion du centrosome, à la manière de son partenaire IFT88 au niveau des MTs mitotiques, ajoutant un autre mécanisme physiopathologique aux ciliopathies associées à IFT52Ciliopathies are multisystemic disorders due to mutations in genes encoding proteins localizing at the cilium. Cilia are microtubule-based organelles present at the surface of almost all vertebrate cells. IFT52 is a key component of the intraflagellar transport IFT-B complex that guarantees cilium formation and functions. Missense, nonsense mutations and in-frame deletions have been identified in IFT52 in three families presenting distinct phenotypes of ciliopathies: multicystic kidney dysplasia (MCKD) or skeletal dysplasia such as short ribs-polydactyly (SRP) or Sensenbrenner (SB). To understand the phenotype variability, we sought to characterize the pathogenicity of the mutations by using patient fibroblasts and CRISPR/Cas9-induced cellular and animal models. First, we showed that the nature of the in-frame (SRP) and nonsense mutations (SB) actually led to a frameshift and an in- frame exon skipping, respectively that partially explained the genotype/phenotype correlation. On the contrary, analyses of missense mutations from SRP and MCDK cases indicated that SRP mutation had a more severe impact on IFT52 function than the DRK mutation, confirming the pathogenicity of the mutations and explaining the skeletal defects of SRP case. However, it did not explain the renal phenotype variability observed amongst the individuals. Thus, we considered a second homozygous missense mutation in the MCDK case in UBE2C, a gene encoding an ubiquitin conjugating enzyme involved in metaphase/anaphase transition. Tridimensionnal modelisation of the protein structure showed that the mutation affected the binding with ubiquitin and likely the function of the protein. In vitro studies confirmed that the mutation delayed mitosis exit. In order to study the role of Ube2c in kidney development, a knock-in (KI) mouse line of the patient mutation was generated by CRISPR/Cas9 technique. Although Ube2cKI/KI failed to thrive and died early, similarly to the knock-out line, kidneys appeared normal in these animals. So we hypothesized thet the renal phenotype of the MCDK case could come from a synergy between the mutations in IFT52 and UBE2C. To test this hypothesis, we performed synergy experiments by injecting ift52 morpholino in zebrafish embryos issued from ube2c+/- incrosses, but we did not observe kideny defects in ube2c-/- embryos. Thus, we conclude that either the MCDK individual present another mutation responsible for the renal phenotype or the animal models we used (mouse, zebrafish) were not appropriate to study the involvement of UBE2C in human kidney development. Surprisingly, the study on IFT52 mutations highlighted an abnormal splitting of the centrioles of the same centrosome in Ift52-/- cells. Centrosome cohesion is guaranteed by two mechanisms: a protein linker between proximal parts of the centrioles and the microtubule (MTs) forces exerted on the centrosome. The proteins from the protein linker, such as c-Nap1 and Rootletin, did not seem to be affected by the loss of Ift52. However, we showed that Ift52 interacted and partially co-localized with centrin at the distal part of the centriole, suggesting a role for Ift52 in the MTs mechanism. Indeed, we showed that in Ift52-/- cells, MTs presented failed to anchor at the centrosome and their dynamic was reduced. These defects could be the cause of the centrosome splitting and suggest an extra-ciliary role for IFT52 in MTs anchoring and centrosome cohesion, as its partner IFT88 in mitosis, adding another physiopathological mechanism to the IFT52-associated ciliopathies
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Hage-Sleiman, Rouba. "Impact of tululin binding cofactor C (TBCC) on microtubule mass and dynamics, cell cycle, tumor growth and response to chemotherapy in breast cancer". Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10085/document.
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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 traitementsThe 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
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Hoover, Ashtyn. "The Role of Small GTPase RhoG in Focal Adhesion Dynamics and Contractility". University of Toledo / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556712457014336.
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Shao, Qiangqiang. "Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules is Required in Netrin- 1-Mediated Axonal Repulsion". University of Toledo / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513185574993639.
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Barlukova, Ayuna. "Dynamic instability of microtubules and effect of microtubule targeting agents". Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0064.
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L'objectif de cette thèse est de proposer des modèles mathématiques permettant de décrire l'instabilité dynamique d'une population de microtubules (MTs) et l'effet de médicaments sur cette instabilité. L'instabilité dynamique des MTs joue un rôle extrêmement important dans les processus de la mitose et de la migration cellulaire et donc dans la progression tumorale. L'instabilité dynamique est un processus complexe qui implique différents états de la tubuline (polymérisée ou non-polymérisée, tubuline-GTP ou tubuline-GDP qui correspondent à deux états énergétiques différents des dimères) et qui résulte de processus chimiques (polymérisation, dépolymérisation, hydrolyse, recyclage, nucléation) liant ces différents états de la tubuline. Décrire cette complexité par le biais de modèles mathématiques permet alors de tester des hypothèses biologiques quant à l'impact de chacun de ces processus et l'action de molécules anti-MTs. De récents travaux suggèrent que le "vieillissement" des MTs impacte leur dynamique. Nous avons testé dans ce travail l'hypothèse que ce "vieillissement" accélère l'hydrolyse du GTP au sein de la tubuline. Nous avons construit de nouveaux modèles couplant deux équations de transport multi-D avec deux équations différentielles ordinaires impliquant des termes intégraux. Nous avons calibrer notre nouveau modèle à partir des données expérimentales; tester l'hypothèse biologique sur le mécanisme du processus de vieillissement; analyser la sensibilité du modèle par rapport aux paramètres décrivant les processus; tester différentes hypothèses quant l'effet des médicaments anti-MTsThe aim of this thesis is to design new mathematical models that are able to appropriately describe dynamic instability of a population of microtubules (MTs) and effect of drugs on MT dynamics. MT dynamic instability play an important role in the processes of mitosis and cell migration and, thus, in cancer progression. Dynamic instability is a complex process that involves different states of tubulin (polymerized or non-polymerized, GTP-tubulin or GDPtubulin that correspond to two different energetic states of tubulin dimers) that resulted from chemical processes (polymerization, depolymerization, hydrolysis, recycling, nucleation) linking these different states of tubulin. Description of this complexity by mathematical models enables one to test biological hypotheses concerning the impact of each process and action of drugs on microtubule dynamics. Recent observations show that MT dynamics depends on aging of MT. One of the aims of the work is to test the hypothesis that MT aging results from the acceleration of the GTP hydrolysis. We construct for that new models that couple two multidimensional transport equations with two ordinary differential equations involving integral terms. We have calibrated our models on the basis of experimental data; tested biological hypothesis on mechanism of aging process; performed a sensitivity analysis of the model with respect to parameters describing chemical processes; and tested hypotheses concerning actions of drugs
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Nam, Gi-moon. "Dynamics of confined biofilaments". Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAE048/document.
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Cette thèse est consacrée à la mécanique et à la mécanique statistique de biofilaments/biopolymères et de leur modèle le plus répandu le Worm-Like Chain (WLC) qu’il s’avère nécessaire d’étendre. Nous étudions WLC à 2-d en présence d’obstacles plus proches que la longueur de persistance. Nous caractérisons le mouvement aux temps courts par des simulations numériques complétées par des calculs analytiques. Des concepts similaires servent à décrire des ADN greffés balayés par le front d’une vésicule en cours d’étalement, l’adhésion de la vésicule est promue par des paires biotine/streptavidine qui contraignent les molécules d'ADN sur des chemins étroits où ils peuvent être imagés. Les microtubules (MT) ici stabilisés au taxol, présentent par contre certains comportements qui échappent au WLC et doivent être ramenés à leur structure interne : i)les déflexions latérales d’un MT attaché par un bout correspondent à une longueur de persistance apparente qui augmente avec la longueur ii) les MT adoptent des formes super-hélicoïdales. Ces deux points sont établis au moyen d’analyses de forme des MT. Des transitions de forme corrélées le long du MT mises en évidence sont compatibles avec un modèle basé sur la bistabilité du dimère de tubuline. Finalement un modèle de chaîne super-hélicoïdale comprenant une courbure et une torsion spontanées élargi le WLC. Confiné à 2-d, HWLC peut adopter un état fondamental circulaire ou sinueux caractérisé par le nombre de points d’inflexion où se concentre la torsion (twist-kink). Dans le cas circulaire, il existe des états métastables proches, à petit nombre de twist-kinks, hyperflexiblesThis PhD is devoted to the mechanics and statistical mechanics of biofilaments and their most widespread model, the Worm-Like Chain (WLC) model, which, as it turns out, needs to be extended. We study the WLC in 2-d in the presence of obstacles closer than their persistence length. We characterize the short time motion by numerical simulations complemented by analytical calculations. Similar concepts serve to describe grafted DNAs swept by the front of a spreading vesicle whose adhesion is promoted by biotin/streptavidin bonds, which constrain the DNAs on narrow paths where they can be imaged. Microtubules (MT), here stabilized by taxol, show features which cannot be rationalized by the WLC and shall be related to their internal structure : i)lateral deflections of a clamped MT correspond to an effective persistence length growing with the MT size ii) MT adopt super-helical shapes. These two points are proven by refined image analysis. We analyze shape transitions correlated along the MT which are compatible with a model based on dimer bi-stability. Finally, a super helical chain model (HWLC) allowing for spontaneous curvature and twist is developed which extends the WLC. When confined to 2-d, the HWLC can adopt a ground state which is circular or wavy with inflection points where twist accumulates, so-called twist-kinks. In the circular case there exist close metastable states, with a small number of twist-kinks, which are hyperflexible
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Cancellieri, C. "BETA-ARRESTIN DEPENDENT REGULATION OF CYTOSKELETON DYNAMICS AND SIGNALLING OF CHEMOKINE RECEPTOR ACKR2". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229565.
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Chemokines promote leukocyte migration through the activation of dedicated G-protein coupled receptors. Beyond conventional chemokine receptors, which directly induce cell migration through heterotrimeric Gαi-mediated signalling events, a set of atypical chemokine receptors (ACKRs) have been described. ACKRs do not activate Gαi-mediated signalling activity, but they are mainly involved in shaping the chemokine gradient. The best characterized member of this family is ACKR2.
ACKR2, previously referred to as D6, is a scavenger receptor that binds with high affinity to 13 inflammatory CC chemokines. The scavenging activity of ACKR2 relies on its intracellular traffic properties. Under homeostatic conditions, ACKR2 is mainly localized in intracellular stores associated with both early Rab4/5-positive and recycling Rab11-positive endosomes. At increasing levels of chemokines, ACKR2 increases plasma membrane abundance through an acceleration in the rate of Rab11-depedent recycling pathway, in order to optimize its chemokine scavenging activity. Here, I demonstrated that the intracellular distribution of ACKR2 is maintained by cytoskeletal dynamics. After chemokine engagement, ACKR2 activate a G-protein-independent and β-arrestin-dependent Rac1-PAK1-LIMK1 signalling cascade to finely regulate the actin cytoskeletal and the microtubules network reorganization, to promote receptor up-regulation and scavenging function. ACKR2 is able to recruit and associates both β-arrestins in basal condition, at membrane and intracellular levels, but only β-arrestin1 is recruited after active ligand stimulation, in order to promote a β-arrestin1-dependent signalling pathway, required for supporting the myosin Vb-dependent ACKR2 up-regulation and scavenging properties.
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Merz, Dale R. Jr. "Molecular simulations uncover the nanomechanics of heat shock protein (70 kDa) & Indentation simulations of microtubules reveal katanin severing insights". University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1583154342504106.
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RULLO, Cinzia. "Synthetic approaches to novel derivatives of natural hemiasterlins and epothilone B as potential anticancer drugs interfering with microtubule dynamics". Doctoral thesis, Università degli studi di Ferrara, 2011. http://hdl.handle.net/11392/2388732.
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Cancer is a disease characterized by uncontrolled growth of a group of cells that eludes physiologic turnover mechanisms.
Conventional cytotoxic therapies of cancer often suffer from a lack of specificity, as they present low therapeutic index and considerable toxicity to healthy organs.
Moreover, many kinds of cancer develop resistance to drugs and that is the most important factor in the failure of many chemotherapic treatments.
Most of the effective anticancer drugs currently administered, as those at the moment in clinical trials are derived from natural products which are considered “lead compounds” in drug discovery process.
Dynamic microtubules are among the most successful targets of anticancer drugs, as they are involved in chromosomes segregation during cell division.
Microtubules-interacting agents can be divided into two different functional classes: from one side compounds which stabilize microtubules and those which inhibit the assembly of tubulin heterodimers.
Hemiasterlins are member of a recently discovered family of natural tripeptides, isolated from marine sponge and containing three highly modified aminoacids which are responsible of their stability and in vivo activity. These compounds disrupt spindle microtubules, inhibit its growth and induce the self-association of tubulin dimers into single-walled rings and spirals.
In order to circumvent the P-glycoprotein-mediated resistance of hemiasterlin, taltubulin (HTI-286, 1), an analogue where a phenyl group replaces the indole ring of hemiasterlin, was recently synthetized thanks to SAR studies.
Based on its interesting biologic profile, we have thought to invert the aromatic ring with the methyl group on nitrogen at N-terminus aminoacid of HTI-286, with the aim to obtain a series of compounds with a biological activity better than parent compound, and easily synthesizable 3.
We exploited the well-known potentiality of silver oxide as promoter in stereoselective nucleophilic substitution reaction of bromo-acylpeptides, affording a small group of tripeptides structurally variable in the perspective of a preliminary biological evaluation.
Some compounds showed potent biological activity as growth inhibitors of some cancer cells lines and tubulin polymerization inhibitors.
A future approach has also been envisaged to overcome the disadvantage related to cancer therapy toxicity, namely the selective delivery of drugs to the tumor site, by the conjugation of a drug with a proper carrier.
Among the microtubules stabilizing agents, epothilones are a promising class of drugs, effective in particular solid tumor. Epothilones contain a 16-membered macrolide ring with a methylthiazole side chain and based on SAR data, not all modification are possible to obtained active compounds.
ZK-EPO B (5), a benzothiazole analogue of EPO B (4), has an interesting biological profile and encouraged by its progress in clinical trials, the group of Prof. Altmann (where I spent 9 months during my Ph. D.) has thought to synthesize an analogue of EPO B (6), provided with a functional handle, at position 22 for antibody conjugation, a further modification was directed to the natural 12,13-epoxide moiety has been replaced by a cyclopropane ring.
This modification eliminates potential problems of chemical and/or metabolic stability linked to the epoxide function; moreover the cyclopropane doesn’t lead to any loss of antiproliferative activity.
The aim of my project was the synthesis of a cyclopropanated intermediate (7), whose desired stereoselectivity was the basis to decide the future of the project, installing at the same time the synthetic route of a heterocyclic core of new EPO B analogs.
It was observed that Charette cyclopropanation of an unsubstituted allylic alcohol in the Northern part of the macrocycle was high selective (90:1), but the presence of a methyl at the double bond (C12) decreased the selectivity. A diastereomeric ratio ~ 20 :1 in favour of the right diastereoisomer was tolerated to carry on the whole synthesis and as regarding my project, the outcome of this reaction was acceptable (dr 15:1).
Another challenge was the aldol reaction to install the chiral centre at position 15 of the macrocycle. This was accomplished thanks to Evans oxazolidinone chemistry.
27
Nam, Gi-Moon. "Dynamics of confined biofilaments". Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00976029.
Texto completoResumen
This PhD is devoted to the mechanics and statistical mechanics of biofilaments and their most widespread model, the Worm-Like Chain (WLC) model, which, as it turns out, needs to be extended. We study the WLC in 2-d in the presence of obstacles closer than their persistence length. We characterize the short time motion by numerical simulations complemented by analytical calculations. Similar concepts serve to describe grafted DNAs swept by the front of a spreading vesicle whose adhesion is promoted by biotin/streptavidin bonds, which constrain the DNAs on narrow paths where they can be imaged. Microtubules (MT), here stabilized by taxol, show features which cannot be rationalized by the WLC and shall be related to their internal structure : i)lateral deflections of a clamped MT correspond to an effective persistence length growing with the MT size ii) MT adopt super-helical shapes. These two points are proven by refined image analysis. We analyze shape transitions correlated along the MT which are compatible with a model based on dimer bi-stability. Finally, a super helical chain model (HWLC) allowing for spontaneous curvature and twist is developed which extends the WLC. When confined to 2-d, the HWLC can adopt a ground state which is circular or wavy with inflection points where twist accumulates, so-called twist-kinks. In the circular case there exist close metastable states, with a small number of twist-kinks, which are hyperflexible.
28
Zumdieck, Alexander. "Dynamics of Active Filament Systems". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1139849910030-68242.
Texto completoResumen
Aktive Filament-Systeme, wie zum Beispiel das Zellskelett, sind Beispiele einer interessanten Klasse neuartiger Materialien, die eine wichtige Rolle in der belebten Natur spielen. Viele wichtige Prozesse in lebenden Zellen wie zum Beispiel die Zellbewegung oder Zellteilung basieren auf dem Zellskelett. Das Zellskelett besteht aus Protein-Filamenten, molekularen Motoren und einer großen Zahl weiterer Proteine, die an die Filamente binden und diese zu einem Netz verbinden können. Die Filamente selber sind semifexible Polymere, typischerweise einige Mikrometer lang und bestehen aus einigen hundert bis tausend Untereinheiten, typischerweise Mono- oder Dimeren. Die Filamente sind strukturell polar, d.h. sie haben eine definierte Richtung, ähnlich einer Ratsche. Diese Polarität begründet unterschiedliche Polymerisierungs- und Depolymerisierungs-Eigenschaften der beiden Filamentenden und legt außerdem die Bewegungsrichtung molekularer Motoren fest. Die Polymerisation von Filamenten sowie Krafterzeugung und Bewegung molekularer Motoren sind aktive Prozesse, die kontinuierlich chemische Energie benötigen. Das Zellskelett ist somit ein aktives Gel, das sich fern vom thermodynamischen Gleichgewicht befindet. In dieser Arbeit präsentieren wir Beschreibungen solcher aktiven Filament-Systeme und wenden sie auf Strukturen an, die eine ähnliche Geometrie wie zellulare Strukturen haben. Beispiele solcher zellularer Strukturen sind Spannungsfasern, kontraktile Ringe oder mitotische Spindeln. Spannungsfasern sind für die Zellbewegung essentiell; sie können kontrahieren und so die Zelle vorwärts bewegen. Die mitotische Spindel trennt Kopien der Erbsubstanz DNS vor der eigentlichen Zellteilung. Der kontraktile Ring schließlich trennt die Zelle am Ende der Zellteilung. In unserer Theorie konzentrieren wir uns auf den Einfluß der Polymerisierung und Depolymerisierung von Filamenten auf die Dynamik dieser Strukturen. Wir zeigen, dass der kontinuierliche Umschlag (d.h. fortwährende Polymerisierung und Depolymerisierung) von Filamenten unabdingbar ist für die kontraktion eines Rings mit konstanter Geschwindigkeit, so wie in Experimenten mit Hefezellen beobachtet. Mit Hilfe einer mikroskopisch motivierten Beschreibung zeigen wir, wie "filament treadmilling", also Filament Polymerisierung an einem Ende mit der gleichen Rate wie Depolymerisierung am anderen Ende, zur Spannung in Filament Bündeln und Ringen beitragen kann. Ein zentrales Ergebnis ist, dass die Depolymerisierung von Filamenten in Anwesenheit von filamentverbindenden Proteinen das Zusammenziehen dieser Bündel sogar in Abwesenheit molekulare Motoren herbeiführen kann. Ferner entwickeln wir eine generische Kontinuumsbeschreibung aktiver Filament-Systeme, die ausschließlich auf Symmetrien der Systeme beruht und von mikroskopischen Details unabhängig ist. Diese Theorie erlaubt uns eine komplementäre Sichtweise auf solche aktiven Filament-Systeme. Sie stellt ein wichtiges Werkzeug dar, um die physikalischen Mechanismen z.B. in Filamentbündeln aber auch bei der Bildung von Filamentringen im Zellkortex zu untersuchen. Schließlich entwickeln wir eine auf einem Kräftegleichgewicht basierende Beschreibung für bipolare Strukturen aktiver Filamente und wenden diese auf die mitotische Spindel an. Wir diskutieren Bedingungen für die Bildung und Stabilität von SpindelnActive filament systems such as the cell cytoskeleton represent an intriguing class of novel materials that play an important role in nature. The cytoskeleton for example provides the mechanical basis for many central processes in living cells, such as cell locomotion or cell division. It consists of protein filaments, molecular motors and a host of related proteins that can bind to and cross-link the filaments. The filaments themselves are semiflexible polymers that are typically several micrometers long and made of several hundreds to thousands of subunits. The filaments are structurally polar, i.e. they possess a directionality. This polarity causes the two distinct filament ends to exhibit different properties regarding polymerization and depolymerization and also defines the direction of movement of molecular motors. Filament polymerization as well as force generation and motion of molecular motors are active processes, that constantly use chemical energy. The cytoskeleton is thus an active gel, far from equilibrium. We present theories of such active filament systems and apply them to geometries reminiscent of structures in living cells such as stress fibers, contractile rings or mitotic spindles. Stress fibers are involved in cell locomotion and propel the cell forward, the mitotic spindle mechanically separates the duplicated sets of chromosomes prior to cell division and the contractile ring cleaves the cell during the final stages of cell division. In our theory, we focus in particular on the role of filament polymerization and depolymerization for the dynamics of these structures. Using a mean field description of active filament systems that is based on the microscopic processes of filaments and motors, we show how filament polymerization and depolymerization contribute to the tension in filament bundles and rings. We especially study filament treadmilling, an ubiquitous process in cells, in which one filament end grows at the same rate as the other one shrinks. A key result is that depolymerization of filaments in the presence of linking proteins can induce bundle contraction even in the absence of molecular motors. We extend this description and apply it to the mitotic spindle. Starting from force balance considerations we discuss conditions for spindle formation and stability. We find that motor binding to filament ends is essential for spindle formation. Furthermore we develop a generic continuum description that is based on symmetry considerations and independent of microscopic details. This theory allows us to present a complementary view on filament bundles, as well as to investigate physical mechanisms behind cell cortex dynamics and ring formation in the two dimensional geometry of a cylinder surface. Finally we present a phenomenological description for the dynamics of contractile rings that is based on the balance of forces generated by active processes in the ring with forces necessary to deform the cell. We find that filament turnover is essential for ring contraction with constant velocities such as observed in experiments with fission yeast
29
Nehlig, Anne. "La protéine ATIP3 et ses partenaires d’interaction : de nouvelles cibles thérapeutiques contre le cancer du sein". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS461.
Texto completoResumen
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 personnalisésBreast 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
30
Kawamura, Eiko. "Mechanisms of microtubule dynamics regulation by the MICROTUBULE ORGANIZATION 1 protein". Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31198.
Texto completoResumen
The Arabidopsis thaliana MOR1 (MICROTUBULE ORGANIZATION1) protein belongs to the MAP215/Dis1 family of microtubule-associated proteins. The temperature-sensitive mor1 mutants have N-terminal amino acid substitutions, which lead to cortical microtubule disorganization (Whittington et al., 2001). Here I demonstrate by use of live cell imaging and immunolabelling that MOR1 is important for function and organization of all microtubule arrays during cell division and keeps microtubules highly dynamic. Although disruption of mitotic and cytokinetic microtubule arrays is not detected in all dividing mor1-1 cells, quantitative analysis identified distinct defects in preprophase bands, spindles and phragmoplasts. In nearly half of dividing mor1-1 cells, preprophase bands are not detected, and those that do form are often disrupted. mor1-1 spindles and phragmoplasts are short and abnormally organized and persist for longer times than in wild-type, leading to aberrant chromosome arrangements, misaligned cell plates and multinucleate cells. Immunofluorescence indicates that the mutant mor1-1[sup L174F] protein remains associated along the full length of all microtubule arrays, in spite of their disorganization. This suggests the N-terminal region altered by the mor1-1 mutation does not regulate the binding of MOR1 to microtubules, but that it instead plays a role in microtubule dynamics. Microtubule dynamics were therefore measured in living leaf cells expressing three microtubule reporter proteins, GFP-TUA, CMV35S ::GFP-EB1 and Pro [sub EB1] ::EB1-GFP. Dynamics analysis indicates that MOR1 promotes constant and rapid growth and shrinkage and prevents pausing of microtubules. Integrating this new information with previous observations showing that MOR1 and its tobacco homologue MAP200 can bind tubulin oligomers (Twell et al., 2002; Hamada et al., 2004), and that XMAP215 speeds up microtubule growth and shrinkage in 40-60nm increments (Kerssemakers et al., 2006), I postulate that MOR1 might promote microtubule growth and shrinkage by adding and removing tubulin oligomers. Consistent with this idea, the N-terminal region of MOR1 consists of 5 TOG domains, which each span the approximate length of one tubulin dimer within a protofilament chain. I define experiments and present preliminary data to test the hypothesis that each MOR1 protein can add or remove up to 5 tubulin dimers at a time.Science, Faculty of
Botany, Department of
Graduate
31
Ivanova, Ekaterina. "Etudes in vivo des malformations du développement cortical associées à des mutations dans le gène TUBG1". Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ091/document.
Texto completoResumen
Des mutations hétérozygotes faux-sens dans le gène de la tubuline gamma TUBG1, ont été identifiées dans le contexte des malformations du développement cortical, associées à une déficience intellectuelle et à l'épilepsie. Ici, nous avons étudié par la technique d’électroporation in-utero et par des études in vivo, l’effet de quatre de ces variantes sur le développement cortical. Nous montrons que les mutations dans TUBG1 affectent le positionnement neuronal dans la plaque corticale, en perturbant la locomotion des neurones nouvellement nés, mais sans affecter la neurogenèse. Nous proposons que la γ-tubuline mutante affecte le fonctionnement global de ses complexes, et en particulier leur rôle dans la régulation de la dynamique des microtubules. De plus, nous avons développé un modèle de souris knock-in Tubg1Y92C/+ et évalué les conséquences de la mutation sur le développement cortical, les caractéristiques neuroanatomiques et le comportement. Les souris mutantes présentent une microcéphalie globale, des anomalies du néocortex et de l'hippocampe, des altérations du comportement et une susceptibilité épileptique. Ainsi, nous montrons que les souris Tubg1Y92C/+ miment au moins partiellement le phénotype humain et représentent donc un modèle pertinent pour d'autres investigations de la physiopathologie des malformations du développement corticalMissense heterozygous variants in the gamma tubulin gene TUBG1 have been linked to malformations of cortical development, associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning within the cortical wall, by a disrupting the locomotion of newly born neurons but without affecting neurogenesis. We propose that mutant γ-tubulin affects overall functioning of γ-tubulin complexes, and in particular their role in the regulation of microtubule dynamics. Additionally, we developed a knock-in Tubg1Y92C/+ model and assessed consequences of the mutation on cortical development, neuroanatomical features and behaviour. Mutant mice present with global microcephaly, neocortical and hippocampal abnormalities, behavioural alterations and epileptic susceptibility. Thus, we show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of malformations of cortical development
32
Chaurasia, S. "IN SILICO STUDY OF PROTEIN PROTEIN INTERACTION STABILIZATION AND MECHANICAL FORCE APPLICATION ON BIOMOLECULES". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229253.
Texto completoResumen
Targeting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies have provided evidences for the development of small molecules modulating protein-protein interactions. In Part I, it is demonstrated that how a small molecule can induce the formation of an otherwise unstable protein-protein complex. A study of the stabilization of a FKBP12-FRB complex by a small molecule rapamycin is presented. The stability of the complex is analyzed and its interactions are characterized at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin. The reported results and the good performance of standard molecular modeling techniques in describing the model system can be interesting not only in the design and development of improved molecules acting as FKBP12–FRB protein interaction stabilizers, but also in the somehow neglected study of protein-protein interactions stabilizers in general.
In Part II, studies regarding computational modeling of the application of mechanical force to biomolecules is presented. This part is further divided into two chapters since the investigations have been performed on two biological systems. In the first chapter of Part II (chapter 6), it is described that how the osmolyte molecules affect the mechanical unfolding of a peptide. The mechanical unfolding of peptide has been performed by using Steered Molecular Dynamics. In this study, the effect of four different osmolytes on the free energy difference between the folded and the denatured state have been calculated. The observed trend mirrors the expected behavior of the studied osmolytes and unfolding pathways analysis allows an insight into the mechanism of action of osmolytes.
After the successful application of Steered molecular dynamics technique on the β-hairpin peptide, the same is applied on tubulin heterodimers for the in-depth study of the lateral and longitudinal interactions which are responsible for the stability and dynamics of the microtubules. In the other chapter of Part II (chapter 7), these interactions are studied with the help of mechanical dissociation of the tubulin heterodimers. These studies have allowed the identification of the critical interactions responsible for the binding of tubulin heterodimers laterally as well as longitudinally. The observations obtained could be important for the design of compounds that target these interactions and acts as microtubule inhibitors or stabilizers.
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Zumdieck, Alexander. "Dynamics of Active Filament Systems: The Role of Filament Polymerization and Depolymerization". Doctoral thesis, Technische Universität Dresden, 2005. https://tud.qucosa.de/id/qucosa%3A24642.
Texto completoResumen
Aktive Filament-Systeme, wie zum Beispiel das Zellskelett, sind Beispiele einer interessanten Klasse neuartiger Materialien, die eine wichtige Rolle in der belebten Natur spielen. Viele wichtige Prozesse in lebenden Zellen wie zum Beispiel die Zellbewegung oder Zellteilung basieren auf dem Zellskelett. Das Zellskelett besteht aus Protein-Filamenten, molekularen Motoren und einer großen Zahl weiterer Proteine, die an die Filamente binden und diese zu einem Netz verbinden können. Die Filamente selber sind semifexible Polymere, typischerweise einige Mikrometer lang und bestehen aus einigen hundert bis tausend Untereinheiten, typischerweise Mono- oder Dimeren. Die Filamente sind strukturell polar, d.h. sie haben eine definierte Richtung, ähnlich einer Ratsche. Diese Polarität begründet unterschiedliche Polymerisierungs- und Depolymerisierungs-Eigenschaften der beiden Filamentenden und legt außerdem die Bewegungsrichtung molekularer Motoren fest. Die Polymerisation von Filamenten sowie Krafterzeugung und Bewegung molekularer Motoren sind aktive Prozesse, die kontinuierlich chemische Energie benötigen. Das Zellskelett ist somit ein aktives Gel, das sich fern vom thermodynamischen Gleichgewicht befindet. In dieser Arbeit präsentieren wir Beschreibungen solcher aktiven Filament-Systeme und wenden sie auf Strukturen an, die eine ähnliche Geometrie wie zellulare Strukturen haben. Beispiele solcher zellularer Strukturen sind Spannungsfasern, kontraktile Ringe oder mitotische Spindeln. Spannungsfasern sind für die Zellbewegung essentiell; sie können kontrahieren und so die Zelle vorwärts bewegen. Die mitotische Spindel trennt Kopien der Erbsubstanz DNS vor der eigentlichen Zellteilung. Der kontraktile Ring schließlich trennt die Zelle am Ende der Zellteilung. In unserer Theorie konzentrieren wir uns auf den Einfluß der Polymerisierung und Depolymerisierung von Filamenten auf die Dynamik dieser Strukturen. Wir zeigen, dass der kontinuierliche Umschlag (d.h. fortwährende Polymerisierung und Depolymerisierung) von Filamenten unabdingbar ist für die kontraktion eines Rings mit konstanter Geschwindigkeit, so wie in Experimenten mit Hefezellen beobachtet. Mit Hilfe einer mikroskopisch motivierten Beschreibung zeigen wir, wie "filament treadmilling", also Filament Polymerisierung an einem Ende mit der gleichen Rate wie Depolymerisierung am anderen Ende, zur Spannung in Filament Bündeln und Ringen beitragen kann. Ein zentrales Ergebnis ist, dass die Depolymerisierung von Filamenten in Anwesenheit von filamentverbindenden Proteinen das Zusammenziehen dieser Bündel sogar in Abwesenheit molekulare Motoren herbeiführen kann. Ferner entwickeln wir eine generische Kontinuumsbeschreibung aktiver Filament-Systeme, die ausschließlich auf Symmetrien der Systeme beruht und von mikroskopischen Details unabhängig ist. Diese Theorie erlaubt uns eine komplementäre Sichtweise auf solche aktiven Filament-Systeme. Sie stellt ein wichtiges Werkzeug dar, um die physikalischen Mechanismen z.B. in Filamentbündeln aber auch bei der Bildung von Filamentringen im Zellkortex zu untersuchen. Schließlich entwickeln wir eine auf einem Kräftegleichgewicht basierende Beschreibung für bipolare Strukturen aktiver Filamente und wenden diese auf die mitotische Spindel an. Wir diskutieren Bedingungen für die Bildung und Stabilität von Spindeln.Active filament systems such as the cell cytoskeleton represent an intriguing class of novel materials that play an important role in nature. The cytoskeleton for example provides the mechanical basis for many central processes in living cells, such as cell locomotion or cell division. It consists of protein filaments, molecular motors and a host of related proteins that can bind to and cross-link the filaments. The filaments themselves are semiflexible polymers that are typically several micrometers long and made of several hundreds to thousands of subunits. The filaments are structurally polar, i.e. they possess a directionality. This polarity causes the two distinct filament ends to exhibit different properties regarding polymerization and depolymerization and also defines the direction of movement of molecular motors. Filament polymerization as well as force generation and motion of molecular motors are active processes, that constantly use chemical energy. The cytoskeleton is thus an active gel, far from equilibrium. We present theories of such active filament systems and apply them to geometries reminiscent of structures in living cells such as stress fibers, contractile rings or mitotic spindles. Stress fibers are involved in cell locomotion and propel the cell forward, the mitotic spindle mechanically separates the duplicated sets of chromosomes prior to cell division and the contractile ring cleaves the cell during the final stages of cell division. In our theory, we focus in particular on the role of filament polymerization and depolymerization for the dynamics of these structures. Using a mean field description of active filament systems that is based on the microscopic processes of filaments and motors, we show how filament polymerization and depolymerization contribute to the tension in filament bundles and rings. We especially study filament treadmilling, an ubiquitous process in cells, in which one filament end grows at the same rate as the other one shrinks. A key result is that depolymerization of filaments in the presence of linking proteins can induce bundle contraction even in the absence of molecular motors. We extend this description and apply it to the mitotic spindle. Starting from force balance considerations we discuss conditions for spindle formation and stability. We find that motor binding to filament ends is essential for spindle formation. Furthermore we develop a generic continuum description that is based on symmetry considerations and independent of microscopic details. This theory allows us to present a complementary view on filament bundles, as well as to investigate physical mechanisms behind cell cortex dynamics and ring formation in the two dimensional geometry of a cylinder surface. Finally we present a phenomenological description for the dynamics of contractile rings that is based on the balance of forces generated by active processes in the ring with forces necessary to deform the cell. We find that filament turnover is essential for ring contraction with constant velocities such as observed in experiments with fission yeast.
34
Su, Xiaolei. "Regulation of Microtubule Dynamics by Molecular Motors". Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10145.
Texto completoResumen
Kinesin superfamily motors have a well-characterized ability to move along microtubules and transport cargo. However, some members of the kinesin superfamily can also remodel microtubule networks by controlling tubulin polymerization dynamics and by organizing microtubule structures. The kinesin-8 family of motors play a central role in cellular microtubule length control and in the regulation of spindle size. These motors move in a highly processive manner along the microtubule lattice towards plus ends. Once at the microtubule plus end, these motors have complex effects on polymerization dynamics: kinesin-8s can either destabilize or stabilize microtubules, depending upon the context. My thesis work identified a tethering mechanism that facilitates the processivity and plus end-binding activity of Kip3 (kinesin-8 in budding yeast), which is essential for the destabilizing activity of kinesin-8 in cells. A concentration-dependent model was proposed to explain the divergent effects of Kip3 on microtubule dynamics. Moreover, a novel activity of Kip3 in organizing microtubules was discovered: Kip3 can slide anti-parallel microtubules apart. The sliding activity of Kip3 counteracts the depolymerizing activity of Kip3 in controlling spindle length and stability. A lack of sliding activity causes fragile spindles during the process of chromosome segregation in anaphase. The tail domain of Kip3, which binds both microtubules and tubulin dimers, plays a critical role in all these activities. Together, my work defined multiple mechanisms by which Kip3 remodels the microtubule cytoskeleton. The physiological importance of these regulatory mechanisms will be discussed.
35
Huang, Huai. "Modulation of Microtubule Dynamics in Netrin Signaling". University of Toledo / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513347576691818.
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Reis, Rita Margarida Duarte Pires dos. "The involvement of the protein Mast in microtubule dynamics and kinetochore-microtubule interactions". Doctoral thesis, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/24551.
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Reis, Rita Margarida Duarte Pires dos. "The involvement of the protein Mast in microtubule dynamics and kinetochore-microtubule interactions". Tese, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/24551.
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Deng, Xian. "Prosthecobacter BtubAB form bacterial mini microtubules". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275719.
Texto completoResumen
The tubulin/FtsZ superfamily contains a large set of proteins that spans through all kingdoms of life, with αβ-tubulins being the eukaryotic representatives and FtsZ being the best studied prokaryotic homologue. It is believed that all tubulin/FtsZ-related proteins have evolved from a common ancestor, however, members from this superfamily have diverged in many aspects. αβ-tubulins polymerise into giant and hollow microtubules in the presence of GTP. Despite the size of around 25 nm wide, microtubules display sophisticated dynamics. In particular, dynamic instability, the stochastic change between fast growth and rapid shrinkage, is a hallmark of microtubules. In contrast to αβ-tubulins, FtsZ lacks the C-terminal domain of tubulins and it probably functions as single homopolymeric protofilaments, possibly through treadmilling dynamics. There is strong divergence of the biological functions in the tubulin/FtsZ superfamily. Microtubules are involved in fundamental processes such as motility, transport and chromosomal segregation, whereas FtsZ is involved in bacterial cytokinesis (bacterial cell division), and the equivalent role of FtsZ is carried out by actin-based and ESCRTIII-based systems in eukaryotes. It seems that there is a big evolutionary gap between αβ-tubulins and FtsZ, and the only properties that are conserved within the tubulin/FtsZ superfamily are fold, protofilament formation and GTPase activity. In 2002, a pair of tubulin-like genes, btuba and btubb were identified in Prosthecobacter bacteria, with higher sequence homology to eukaryotic tubulins than FtsZ or any other bacterial homologues. The crystal structures solved later revealed, again, a closer similarity to αβ-tubulins than to their prokaryotic equivalents. It has been known for a while that BtubAB form filaments in the presence of GTP, however, little knowledge has been available regarding the filament architecture. In this project, I determined the near atomic resolution structure of the in vitro BtubAB filament using cryoEM and cryoET, revealing a hollow tube that consists of four protofilaments. A closer look showed that BtubAB filaments have many conserved microtubule features including: an overall polarity, similar longitudinal contacts, M-loops in lateral interfaces, and the presence of the seam, a structural hallmark of microtubules. My study also shows that BtubC, a protein with a TPR fold, binds to the BtubAB filaments in a stoichiometric manner, similar to some MAPs on microtubules. Based on this work, I concluded that BtubAB from Prosthecobacter form bacterial ‘mini microtubules’, and my work provided interesting insight into the evolution of tubulin/FtsZ-related proteins.
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Rookyard, Chris. "Modelling and image processing of microtubule dynamics and organisation". Thesis, University of East Anglia, 2014. https://ueaeprints.uea.ac.uk/57213/.
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Microtubules, dynamic protein polymers, form networks that are essential for intracellular organisation. Involved in many cellular processes that are vital in development and homeostasis, improper regulation of the microtubule network is implicated in various diseases. This work addresses the relationships between microtubule dynamics and organisation, using image processing and modelling, focussing on two features of microtubule organisation: radiality and alignment. The hypothesis that radiality results from modulation of dynamics at the cell periphery was tested. Firstly, cells in which the small GTPase Rac1 was inhibited were used as a model for perturbed radiality. Measurements of microtubule dynamics in central and peripheral regions showed that Rac1 inhibition alters microtubule dynamics and the orientation of their growth at the cell periphery. Further investigation was carried out with a simple 1-dimensional, two-area dynamics model, which confirmed that a two-area dynamics system is sufficient to target microtubules to a given length. The propensity to grow of any given dynamics parameters is a major determinant of the accuracy of length targeting, while the extent of pausing and the average length have a modulatory effect on accuracy. Simulation of measured dynamics indicated that two-area dynamics may contribute to radiality in reality, but that this mechanism may work in concert with other cortex-specific processes. The alignment of microtubules was quantified with a new application of the Fourier transform. Depletion of +TIP protein EB2 produced highlyaligned microtubules, and inhibition of formins rescued this phenotype. Inhibition of Rac1 produced less-aligned microtubules in otherwise unperturbed cells, while in EB2-depleted cells, microtubules were further aligned. The method was also used to quantify alignment in plant microtubule arrays. This work presents a set of analyses that test ideas as to how the microtubule network is organised, and highlight interesting relationships between dynamics and organisation that will yield exciting future investigation.
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Laguillo, Diego Alejandra 1991. "K-fiber dynamics: a focus on the microtubule minus-ends". Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/671237.
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During cell division, microtubules (MTs) organize into a bipolar spindle that drives faithful chromosome segregation through the kinetochore fibers (k-fibers), bundles of parallel MTs that attach the chromosomes through their plus-ends to the spindle poles through their minus-ends. K-fiber MT plus and minus ends dynamics are coordinated to provide stability at the same time as support error correction, chromosome alignment and segregation. However, k-fiber minus-ends have been poorly characterized.
Electron tomography studies showed that the k-fibers MT minus ends have mixed open or close conformations suggesting complex regulatory mechanisms. Consistently, the silencing of MCRS1, currently the only potential regulator, results in k-fibers with less MTs with an increased proportion of open ends morphologies. TIRF based in vitro reconstitution assays showed that MCRS1 and KANSL3 associate preferentially with one MT end. Another member of the complex, KANSL1 may mediate their interaction formation of a ternary complex. Altogether, my results suggest that the MCRS1-KANSL-complex could dynamically “cap” some K-fiber MT-minus-ends to regulate their depolymerization rates for proper cell division.Durant la divisió cel.lular els microtúbuls (MTs) s’organitzen en el fus mitòtic, que és l’encarregat de la segregació dels cromosomes a través de les anomenades “fibres del cinetocor” (fibres-k). Aquestes fibres son feixos de MTs que connecten a través del seu extrem-(+) els cromosomes amb els pols del fus mitòtic, on tenen el seu extrem-(-). La dinàmica d’aquestes fibres-k en tots dos extrems està coordinada per garantir l’estabilitat del fus mitòtic alhora que permet l’alineament dels cromosomes, la seva segregació i la correcció de posibles errors. Malgrat tot, la dinàmica a l’extrem-(-) gairebé no s’ha caracteritzat. Els nostres estudis amb tomografia electrònica demostren que els extrems-(-) dels MTs de les fibres-k presenten una barreja en les conformacions dels seus extrems obertes i tancades, el que suggereix que estan subjectes a mecanismes de regulació complexos. D’acord amb això, el silenciament de MCRS1, actualment l’únic regulador potencial de la dinàmica a l’extrem-(-), té com a resultat fibres-k amb menys MTs i amb un increment en la proporció d’extrems amb conformacions obertes. Assaigs de reconstitució in vitro basats en microscopia TIRF mostren que MCRS1 i KANSL3 s’uneixen preferentment a un dels extrems del MT. Un altre membre del mateix complex, KANSL1 podria també interaccionar per formar un complex ternari. En conjunt, els meus resultats suggereixen que el complex MCRS1-KANSL podria bloquejar dinàmicament alguns extrems (-) de les fibres-k per tal de regular la seva despolimerizació de manera que la divisió cel.lular sigui adequada.
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Neukirchen, Dorothee. "CLIPs regulate neuronal polarization through microtubule and growth cone dynamics". Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-126639.
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Faller, Elliott M. "Modulation of microtuble dynamics by the microtubule-associated protein MAP1a". Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26371.
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Structural microtubule-associated proteins (MAPs) are capable of interacting with tubulin dimers to regulate the various dynamic stages of microtubules. MAP1a is predominant in the neuronal cell body, axons and dendrites of mature neurons. MAP1 a has been shown to bind microtubules to promote microtubule assembly in vitro. The MAP1a heavy chain molecule is associated with three light chains. The heavy chain, and all three light chains appear to associate with microtubules independent of each other. The purpose of this project was to measure the impact of myc-tagged MAP1 a fragments and myc-tagged light chains associated with MAP1a on microtubule dynamic phases in vivo. Cells from an epithelial kidney cell line (LLCPK1) that had been permanently transfected with human GFP-alpha tubulin were transiently transfected with myc tagged MAP1a heavy and light chain fragments. Cells expressing MAP1a and light chain fragments were used to make direct observations of microtubule dynamics in living cells using fluorescence microscopy. Microtubule ends were photographed at 4-second intervals using a digital camera over a 2-minute duration. All truncated MAP1 a heavy chain fragments that contained the microtubule-binding domain were shown to associate with microtubules. MAP1a fragments containing portions of the projection domain promoted growth and stability of microtubules. Truncated fragments containing different regions of the projection domain of MAP1a demonstrated variations in their impact on microtubule dynamic events by promoting growth or inhibition of shortening phases. Similar to full length MAP1a, LC3 also appeared to promote microtubule growth and stability. Results from the present study suggest that MAP1a and LC3 promote slow, stable growth of microtubules. This type of growth may be important in the maintenance and restructuring of adult neurons.
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Titos, Vivancos Iris 1986. "Topoisomerase II and dynamic microtubules solve sister chromatid intertwinings in anaphase". Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/287225.
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At the metaphase-to-anaphase transition, spindle microtubules pull replicated
chromosomes to the daughter cells, but full separation of long chromosome
arms is achieved in late anaphase. We have created an allelic series of long
chromosomes to elucidate the mechanisms involved in long chromosome
resolution during mitosis. With this method we have shown that long
chromosome cells are sensitized to the loss of genes involved in chromosome
structure and segregation. We have discovered that Topoisomerase II is
needed during anaphase to resolve distal regions of long chromosomes and
that the activity of the microtubule polymerase Stu2 is crucial in the resolution
of catenations. Moreover, we have identified the nuclear organization as a
new source that contributes to the topological stress accumulated in
chromosomes. Thus, topological constraints imposed by chromosome length
and nuclear architecture determine the amount of sister chromatid
intertwinings that must be resolved by Topoisomerase II and dynamic
microtubules during anaphase.A la transició entre metafase i anafase els microtúbuls del fus mitòtic transporten els cromosomes a les cèl·lules filles, tot i això la separació completa dels braços dels cromosomes no succeeix fins al final dʼanafase. Amb lʼobjectiu dʼentendre com es resolen els cromosomes llargs durant anafase, hem creat una sèrie al·lèlica de cromosomes artificalment llargs. Amb aquesta metodologia hem demostrat que les cèl·lules que contenen cromosomes llargs estan sensibilitzades a la pèrdua de gens involucrats en lʼestructura i la segregació de cromosomes. Hem descobert que la Topoisomerasa II es necesària durant anafase per resoldre les regions distals de cromosomes llargs i que lʼactivitat de la polimerasa de microtúbuls, Stu2, és essencial en la resolució de concatenacions entre cromàtides germanes. A més, hem pogut identificar lʼorganització nuclear com una nova font que contribueix a lʼestrés topològic acumulat als cromosomes. En conclusió, les restriccions topològiques que imposen tant la longitud dels cromosomes com lʼarquitectura nuclear determinen la quantitat de concatenacions entre cromàtides germanes que han de ser resoltes per la Topoisomerasa II i els microtúbuls dinàmics durant anafase.
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Campbell, Robert David James. "Information processing in microtubules". Thesis, Queensland University of Technology, 2002.
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Seggio, Maxime. "Etude in vitro des effets de la protéine MAP6 sur le cytosquelette". Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV063/document.
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Le cytosquelette d'une cellule eucaryote est constitué de trois types de polymères différents qui sont l'actine, les filaments intermédiaires et les microtubules. Ces éléments confèrent à la cellule l'essentiel de ses propriétés mécaniques telles que le maintien de l'architecture ou la modification de sa forme pour permettre le déplacement cellulaire. Ils sont également impliqués dans le transport d'organites ou de nutriments d'un bout à l'autre de la cellule, dans la ségrégation des chromosomes lors de la mitose ou encore dans le processus de division cellulaire. Pour répondre aux différents besoins de la cellule, ces filaments sont extrêmement dynamiques et peuvent se désassembler pour se réassembler à un autre endroit de la cellule. Cette dynamicité est régulée par de nombreuses protéines accessoires qui vont être capables de modifier les propriétés intrinsèques des différents filaments (dynamique, mécanique et organisatrice). Parmi ces protéines régulatrices, l'on distingue tout particulièrement les MAPs, pour Microtubule Associated Proteins, capables de modifier la dynamique et la structure des microtubules. MAP6, ou encore STOP pour Stable Tubule Only Peptide, est une MAP neuronale qui fut initialement décrite pour sa capacité à protéger les microtubules d'une exposition au froid ou encore de drogues dépolymérisantes comme le nocodazole. Des souris délétées pour le gène MAP6 montrent des troubles cognitifs et comportementaux proches des patients atteints de schizophrénie, impliquant au moins en partie des défauts de stabilisation des microtubules. Cependant, les effets de la protéine sur les microtubules restaient encore à déterminer. Dans ce contexte, à l'aide de diverses approches biochimiques et vidéomicroscopiques, nous avons montré que la protéine MAP6 est capable d’interagir de façon directe avec les microtubules in vitro et permet leur stabilisation. Elle permet aussi de réguler la dynamique des microtubules en augmentant la vitesse de polymérisation de l'extrémité (+), de diminuer la fréquence de catastrophe et l'apparition d’événements de sauvetage, de façon similaire à d'autres MAPs comme Tau ou MAP2. Cependant, contrairement aux autres MAPs, nous avons montré que MAP6 présente une dualité d'action sur le bout (-) des microtubules en diminuant et figeant très rapidement la dynamique de cette extrémité. Cette dualité pourrait ainsi conférer à MAP6 un rôle essentiel de nucléateur de microtubules en figeant l'extrémité (-) du microtubule et en favorisant la polymérisation et la stabilisation de l'extrémité (+). De plus, la protéine MAP6 est capable de modifier fortement la structure des microtubules. De part leur composition et leur rôle, les microtubules sont les éléments les plus rigides du cytosquelette et forment naturellement un tube creux linéaire. Or en présence de MAP6, les microtubules perdent cet aspect linéaire et adoptent une structure hélicoïdale (avec un pas d'environ 4,5 μm et une hauteur d'environ 1 μm) qui n'avait encore jamais été observée jusqu'à présent. La présence d'une telle population de microtubules dans la cellule pourrait ainsi apporter une certaine résistance mécanique ou encore permettre le maintien de l'architecture de l'axone. Enfin, nous avons montré que MAP6 peut aussi interagir de façon directe avec les filaments d'actines et les associer entre eux pour former des faisceaux. Dans les neurones, de nombreuses molécules ont été identifiées comme étant des régulateurs clés dans le « crosstalk » entre les filaments d'actines et les microtubules. L'interaction et la coordination entre les différents éléments du cytosquelette jouent un rôle essentiel dans la transmission et le relais du message synaptique. MAP6 pourrait être importante pour l'ensemble de ces mécanismes ce qui expliquerait les défauts de plasticité synaptique ainsi que les défauts cognitifs observés chez les souris KO MAP6The eukaryotic cell's cytoskeleton is constitued by three types of different polymers which are the actin filaments, the intermediate filaments and the microtubules. These elements confer on the cell the main part of its mechanical properties such as the architecture preservation or the modification of its shape to allow the cellular movement. They are also involved in the organelles or nutrients transport throughout the cell, in the chromosomes segregation during mitosis or still in the cellular division process. To answer the cell's various needs, these filaments are extremly dynamics and are able to dis-assemblate to re-assemblate in another place of the cell. Tis dynamic is regulated ny numerous proteins which are going to be capable of modifiying the intrinsic properties of the different filaments (dynamic, mechanic and structure). Among them are present the MAPs, for Microtubule-Associated Proteins, which will be able to influence the microtubule dynamics and structure. MAP6, also known as STOP for Stable Tubule Only Peptide, is a neuronal MAP which was initially described for its capacity to protect microtubule from cold or nocodazole exposure. KO MAP6 mice display cognitive and behavioral disorders close to patient with schyzophrenia, involving at least partially microtubules stabilization defects. However, the effects of the protein on the microtubules still remained to determine. In this context, using diverse biochemical and cideomicroscopy technics, we showed that MAP6 is able to directly interact in vitro with the microtubules and stabilizes them. It also regulates the microtubule dynamics by increasing the microtubule growth rate of the plus end extremity, decreases the shrinkage frequency and allows rescue of shrinking microtubules, similarly to other MAPs like Tau or MAP2. However, contrary to the other MAPs, we showed that MAP6 has another effect on the microtubule (-) end by decreazing and freezing its dynamics. This dual effect could confer to MAP6 an essential role of microtubules nucleation by stabilizing the new formed microtubule (-) end and by stabilizing and increasing the (+) end microtubule growth rate. Furthermore, MAP6 is also able to strongly modify the microtubule structure. Microtubules are the stiffest elements of the cytoskeleton and naturally form due to their composition linear hollow tubes. Yet in presence of MAP6, microtubules lose their usual shape and adopt a helical structure (4,5 μm pitch and approximatly 1 μm thickness) which had never been observed until now. The presence of such a population of microtubules in the neuron could thus provide a mechanical strength and allow the preservation of the axon architecture. Finally, we showed that MAP6 can also directly interact with the actin filaments to associate them and form bundles. In neurons, several molecules have been identified as key regulators in the " crosstalk " between actin filaments and microtubules. The interaction and coordination between the different cytoskeletal elements play a vital role in the synaptic transmission. MAP6 may be important for all these mechanisms which would explain the synaptic plasticity and cognitive defects observed in KO MAP6 mice
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Kulkarni, Sarang V. "Microtubule dynamics during early neural differentiation of P19 embryonal carcinoma cells". Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/7613.
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Altered microtubule (MT) stability prior to neurite extension was examined in P19 embryonal carcinoma cells induced to differentiate along the neural pathway by retinoic acid. In undifferentiated P19 cells few acetylated MTs were detected by immunofluorescence staining and these were resistant to disassembly by colchicine at 1 $\mu$g/mL for 45 minutes. Twenty-four hours after neural induction there was an increase in the number of acetylated MTs and in the number of MTs that were resistant to colchicine treatment. This suggested that the MT array in undifferentiated cells is initially dynamic and becomes less dynamic during early neural differentiation prior to morphogenesis. Quantitative immunoblotting showed no change in the levels of total tubulin, but a two-fold increase in the relative level of acetylated tubulin was detected. MT turnover in P19 cells was then examined directly by microinjecting biotin-conjugated bovine brain tubulin. Computer-aided silicon intensified target camera (SIT) imaging of biotin-tubulin incorporation into the MT array showed that by 30 minutes, 92.4 $\pm$ 3.02% of total MTs turned over in undifferentiated cells. In contrast, in the retinoic acid-induced cells 52.8 $\pm$ 19.37% of the MT array turned over in 30 minutes. In conclusion, there are changes in colchicine stability and in tubulin acetylation that are correlated with changes in MT dynamics occurring prior to neurite outgrowth.
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Hussmann, Frauke. "Regulation of S. pombe microtubule dynamics by Alp14, Alp7 and Dis1". Thesis, Institute of Cancer Research (University Of London), 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.551112.
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TOG-family polymerases track microtubule plus ends and modulate dynamics in vivo. In vitro XMAP215 and Stu2 have been shown to exhibit opposing activities, which led to conflicting proposals for molecular mechanisms. To understand more about these apparently contradictory findings and to test candidate models in more detail, I used an in vitro system in which all the components derived from the same organism, Sacchammyce pombe. Deletion mutants of Alp14 and Dis1, the XMAP215 orthologues in S. pombe, show defects in microtubule assembly, consistent with an effect on microtubule dynamics. I have expressed and purified Alp14 and Disl and the TACC-protein Alp7 using a baculovirus system and reconstituted their activities in vitro using dynamically unstable single micro tubules built from purified single isoform (cd and (3)) S. pombe tubulin. In this work I show that Alp14 accelerates the shrinkage of GMP-CPP stabilized microtubules 2x while it accelerates the assembly of GTP-S. pombe tubulin into micro- tubules J Ox. Growth rates change within a growth phase, corresponding to different amounts of Alp14 at the tip. The growth rate acceleration by Alp14 causes a decrease in catastrophe frequency consistent with the extension of the GTP-cap. Remark- ably, although Alp14 binds to mammalian brain tubulin, it does not accelerate the growth rate of mammalian brain micro tubules, and instead is competitively inhibited by 10% mammalian brain tubulin, indicating a catalytic mechanism for Alp14. Catalytic activity and tip tracking are tightly coupled, but separable. Alp14 loses its tip tracking ability upon the addition of 10% mammalian brain tubulin. The TACC-protein Alp7 can restore the tip-tracking ability of Alp14 in the presence of mammalian brain tubulin but not its activity, consistent with reports that Alp7 is a localization factor of AJp14 in vivo. On dynamic S. pombe microtubules Alp7 increases the processivity of Alp14 and suppresses the catastrophe frequency. Growth rates are not further elevated by the addition of Alp7. In contrast, Disl has no TACC-protein interacting partner. Like Alp14 it disassembles GMP-CPP microtubules and increases the growth rate of S. pombe microtubules. Likewise it binds to mammalian brain tubulin without the ability to increase the growth rate of mammalian brain microtubules. However, unlike Alp14, Dis1 is able to tip-track on dynamic mammalian brain microtubules. As both Alp14 and Disl function as S. pombe microtubule polymerases, but not as mammalian brain microtubule polymerases, I concluded that not only the association of tubulin to XMA P215-family members is important for proper function but also that the ready dissociation of tubulin is just as important. Slow dissociation inhibits the activity of XMAP215-family member, supporting a catalytic model. Furthermore I found that higher affinity to the microtubule lattice did not enhance the catalytic activity of Alp14. Instead the higher affinity to the microtubule lattice of Alp14 in the presence of Alp7 increased the residence time of Alp14 at the tip. In return this decreased the catastrophe frequency, enhancing the effectiveness of Alp14 as a polymerase at the microtubule tip. Disl possesses an intrinsic high affinity to the microtubule lattice, tracking the microtubule end readily, but does not exhibit higher polymerase activity than Alp14. On the basis of these data, I propose an assembler model for the molecular mechanism of Alp14-Alp7 and Dis l , in which both XMAP215- family members enhance the growth rate of S. pombe microtubules by accelerating the exchange reaction of GTP-tubulin. High processivity and in return high effectiveness of the catalysts are mediated by Alp7 in the case of Alp14 and by internal microtubule lattice binding regions in the case of Disl.
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Tramontozzi, Peter J. "Microtubule Dynamics During Sperm Aster Centration in Fertilized Sea Urchin Cells". Thesis, Boston College, 2018. http://hdl.handle.net/2345/bc-ir:108018.
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Thesis advisor: David R. BurgessCentration of the nucleus after fertilization is an essential step for setting-up cell division and proper embryonic development in many proliferating cells such as the sea urchin. The sperm aster must capture the female pronucleus for fusion as well as the nucleus becoming positioned at the center of the cell. Microtubules (MTs) are known to play a role in this centration but the exact mechanism remains unknown. This begins to investigate current models of nuclear centration and the role of various interactions. Three phases of migration were observed as the male aster migrated with support in independent movements of the male and female pronuclei. Dimpling affects present that altered the morphology of the cell were observed when engagement occurred between the male and female pronuclei. It was discovered that this dimpling effect was a result of an interaction between MTs and the cortex, as confirmed by visualization of sheared cells in which only the cortex remained. Stemming from previous and current research in the lab, the role of post-translational modifications (PMTs) in nuclear centration was investigated for the different forces exerted due to various factors. Tyrosinated and detyrosinated populations were observed with and without the presence of parthenolide (PTL), an agent that inhibits detyrosination. PTL was observed to not only prevent the proper migration, but also that it expanded tyrosination of tubulin – which would further disrupt the force vectors created through the PMTs promotion of dyneins and kinesins. The results have lead to a new hypothesis to be furthered in order to gain an in-depth understanding in the mechanism(s) for pronuclear migration
Thesis (BS) — Boston College, 2018
Submitted to: Boston College. College of Arts and Sciences
Discipline: Departmental Honors
Discipline: Biology
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Shojania, Feizabadi Mitra. "Physical Concepts of Copolymerization of Microtubules in the Presence of Anti-mitotic Agents". Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/27795.
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A mathematical approach to the concepts of copolymerization of microtubules in the presence of anti-mitotic drugs is presented in this work. A general feature of the mathematical equations is presented. The possibility of having analytical steady state solutions of dynamic equations is investigated. The structure of equations is narrowed down for the specific brand of anti-mitotic drug, colchicine. The behavior of total T-tubulin concentration in the steady state in a regeneration system is investigated and analyzed through the numerical calculations.Ph. D.
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Gilet, Johan. "Dérégulations neuro-développementales impliquées dans les malformations du développement cortical associées aux mutations du gène KIF2A : apport d'un modèle murin knock-in conditionnel". Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ104.
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A l’aide d’études génétiques, notre équipe, a identifié chez des patients avec des malformations du développement cortical (MDC), des mutations dans le gène KIF2A, une kinésine impliquée dans la dynamique des microtubules. Afin d’étudier dans un contexte physiologique l’impact de ces mutations sur le développement cortical, nous avons développé un modèle murin exprimant la mutation p.His321Asp de KIF2A. Les analyses neuro-anatomiques et neuro-développementales des souris exprimant la mutation ont permis de mettre en évidence une microcéphalie, et des anomalies de positionnement neuronale dans le cortex et l’hippocampe. Les explorations phénotypiques, nous ont permis de montrer une susceptibilité accrue à l’épilepsie chez la souris mutante. De plus, des analyses fonctionnelles sur les fibroblastes de patient et par purification de la protéine mutante ont montré l’incapacité de la protéine mutante à dépolymériser les microtubules. Nous pensons que l’ensemble des résultats obtenus lors de ce projet de thèse pourra apporter une meilleure compréhension des mécanismes physiopathologiques impliqués dans les MDC lié aux mutations dans KIF2ABy using genetic studies, our team have identified in patient with malformations of cortical development, missense mutations in the KIF2A gene, a kinesin involved in microtubules depolymerization. In order to study in a physiological context the impact of these mutations on the cortical development, we have developed expressing the KIF2A p.His321Asp mutation. The first neuro-anatomical and neuro-developmental analyzes of the mice expressing the mutation during embryonic development allowed us to highlight microcephaly and neuronal positioning abnormalities in the cortex and the hippocampus. Phenotypic explorations allowed us to highlight increased susceptibility to epilepsy in the mutant mouse. In addition, functional analyzes using patient fibroblasts and purification of the mutant protein have shown that the mutant protein can not depolymerize microtubules. We believe that all the results obtained during this thesis project will provide a better understanding of the pathophysiologic mechanisms involved in malformations of cortical development related to mutations in the KIF2A gene