Dissertations / Theses: 'Microtubules. Mitosis'

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Gallaud, Emmanuel. "Caractérisation du rôle d'Ensconsine / MAP7 dans la dynamique des microtubules et des centrosomes." Thesis, Rennes 1, 2014. http://www.theses.fr/2014REN1S004/document.

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La mitose est une étape essentielle du cycle cellulaire à l’issue de laquelle le génome répliqué de la cellule mère est ségrégé de façon équitable entre les deux cellules filles. Pour cela, la cellule assemble une structure hautement dynamique et composée de microtubules, appelée le fuseau mitotique. En plus d’assurer la bonne ségrégation des chromosomes, le fuseau mitotique détermine l’axe de division, un phénomène particulièrement important pour la division asymétrique où des déterminants d’identité cellulaire doivent être distribués de façon inéquitable entre les deux cellules filles. L’assemblage et la dynamique de ce fuseau sont finement régulés par de nombreuses protéines qui sont associées aux microtubules. Au cour de ma thèse, nous avons identifié 855 protéines constituant l’interactome des microtubules de l’embryon de Drosophile par spectrométrie de masse puis criblé par ARNi 96 gènes peu caractérisés pour un rôle en mitose dans le système nerveux central larvaire. Par cette approche, nous avons identifié 18 candidats sur la base de leur interaction aux microtubules et de leur phénotype mitotique, dont Ensconsine/MAP7. Nous avons montré qu’Ensconsine est capable de s’associer aux microtubules du fuseau et favorise leur polymérisation. De plus, les neuroblastes des larves mutantes présentent des fuseaux raccourcis et une durée de mitose prolongée. Ce délai en mitose est dû à une activation prolongée du point de contrôle du fuseau mitotique qui est essentiel pour une ségrégation correcte des chromosomes en l’absence d’Ensconsine. D’autres part, en association avec la Kinésine-1, son partenaire fonctionnel en interphase, nous avons montré qu’Ensconsine est également impliquée dans la séparation des centrosomes au cours de l’interphase. Ceci entraine une distribution aléatoire des centrosomes pères et fils dans cellules filles. Grâce à cette étude, nous avons révélé deux nouvelles fonctions pour Ensconsine : elle favorise la polymérisation des microtubules et participe donc à l’assemblage du fuseau mitotique et est impliquée, avec la Kinésine-1 dans la dynamique des centrosomes
Mitosis is a key step of the cell cycle that allows the mother cell to segregate its replicated genome equally into the two daughter cells. To do so, the cell assembles a highly dynamic structure composed of microtubules called the mitotic spindle. Additionally to its role in the faithful segregation of chromosomes, the mitotic spindle defines the axis of cell division. This phenomenon is particularly important for the asymmetric cell division in which cell fate determinants have to be unequally distributed between the two daughter cells. Spindle assembly and dynamics are subtly regulated by numerous microtubules-associated proteins. During my PhD, we identified using mass spectrometry, 855 proteins establishing the Drosophila embryo microtubule interactome. An RNAi screen was performed in the larval central nervous system for 96 poorly described genes, in order to identify new mitotic regulators. Based on microtubule interaction and mitotic phenotype, among 18 candidates we focused on Ensconsin/MAP7. We have shown that Ensconsin is associated with spindle microtubules and promotes their polymerization. Neuroblasts from mutant larvae display shorter spindles and a longer mitosis duration. This mitotic delay is a consequence of an extended activation of the spindle assembly checkpoint, which is essential for the proper chromosome segregation in the absence of Ensconsin. This study also showed that, in association with its interphase partner Kinesin-1, Ensconsin is involved in centrosome separation during interphase. As a result, mother and daughter centrosomes are randomly distributed between the daughter cells. In conclusion, we highlighted two news functions of Ensconsin : first, this protein promotes microtubule polymerization and is involved in spindle assembly ; second, Ensconsin and its partner Kinesin-1 regulate centrosome dynamics

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Barton, Richard Christopher. "Microtubules, mitosis and chromosome segregation in Candida albicans." Thesis, University of Kent, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.256985.

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Bouguenina, Mohammed El Habib. "La protéine SMYLE (Short MYomegalin Like EB1 binding protein) dans l'organisation d'un complexe centrosomal, la régulation de la nucléation et la stabilisation des microtubules : conséquences sur la migration et la division des cellules cancéreuses." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM5060.

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Les microtubules (MT) sont des polymères dynamiques ancrés par leurs extrémités moins aux centres de nucléation alors que leurs extrémités plus, explorent le cytoplasme, jusqu’à être stabilisées. Cette capture des extrémités permet l’organisation du réseau des MT. Les +TIP sont un groupe de protéines qui s’associent aux bouts plus des MT. EB1 est une protéine centrale dans le réseau des +TIP qui régule la dynamique des MT et leur interaction avec les structures d’ancrage des extrémités plus. Par protéomique ciblée, nous avons caractérisé l’interactome d’EB1, et mis en évidence un groupe de protéines, précédemment associées aux centres de nucléation incluant AKAP9, une protéine échafaudage pour les protéines kinases A (PKA), la protéine de la matrice péricentriolaire CDK5RAP2, et une isoforme courte de la myomégaline que nous avons appelé SMYLE (Short MYomegalin Like EB1 binding protein). La cartographie moléculaire a permis de montrer que ces protéines formaient un complexe organisé de manière hiérarchique. Nous avons observé que l’association transitoire deLa protéine SMYLE (Short MYomegalin Like EB1 binding protein )dans l'organisation d'un complexe centrosomal, la régulation de la nucléation et la stabilisation des microtubules : conséquences sur la migration et la division des cellules cancéreuses avec les MT néo-nucléés au centrosome favorisait la nucléation et l’acétylation des MT. De manière notable, la déplétion de SMYLE aboutissait à un défaut de nucléation, mais aussi de la capture corticale des MT. Ces défauts dans l’organisation des MT étaient associés à une baisse notable de la migration des cellules de carcinome mammaire et à des anomalies mitotiques. Nos résultats nous permettent de proposer que SMYLE fait partie d’un complexe centrosomale, qui favorise l’assemblage ou la stabilité des microtubules néo-nucléés, contribuant ainsi à des processus majeurs pour le développement tumoral
Microtubules (MT) are dynamic polymers anchored by their minus ends at the MT organizing centers while their highly dynamic plus end explores the cytoplasm until it get stabilized. This plus end capture allows the organization of the MT network. +TIPs are a group of proteins that share the commonality to associate either directly or indirectly to MT plus ends. EB1 is a central protein of the +TIP network that regulates MT dynamics and their interactions with plus end anchoring structures. Using targeted proteomics, we have characterized the EB1 interactome and revealed a set of protein previously shown to associate with the nucleating centers that included AKAP9 an anchoring protein for protein kinase A (PKA), the pericentriolar matrix protein CDK5RAP2 and a short Myomegalin isoform that we named SMYLE (Short MYomegalin Like EB1 binding protein). Molecular mapping revealed that the proteins formed a hierarchically organized complex. We have observed that the transient association of SMYLE to the newly nucleated MTs at the centrosome favored the nucleation and acetylation. Interestingly, SMYLE depletion led to MT nucleation defects, but also a disruption of cortical MT capture. These defects in the MT network were associated with a steep fall in the migratory potential of breast cancer cells and mitotic abnormalities. Our results allow proposing that SMYLE belongs to centrosomal supramolecular complex that favors the assembly and stability of newly nucleated MTs, thus contributing to major processes in tumor development

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Vasileva, Vanya. "Kinetochore-derived microtubules : from molecular regulation to their role in mitosis." Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/7fc60a5c-2fc1-43ef-87ca-4dc20de2200b.

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To maintain genetic integrity in cell division, replicated chromosomes must be segregated accurately into newly formed daughter cells. The faithful segregation of sister chromatids is a crucial event in cell proliferation since it ensures maintenance of a stable set of chromosomes that is critical for the genetic integrity of the daughter cells. Mistakes in sister chromatid segregation have been related to chromosome instability and aneuploidy characteristic of a variety of human diseases, such as cancers and congenital anomalies. During cell division, spindle-pole microtubules (MTs) must capture kinetochores (KTs) so that the chromosomes can be loaded on the mitotic spindle. However, it remains a mystery how spindle-pole MTs can locate KTs with high efficiency, within realistic capture times (Wollman et al. 2005). The appearance of MTs at KTs is correlated with their capture, which is consistent with KT-derived MTs facilitating the initial encounter of KTs by spindle-pole MTs (Kitamura et al. 2010). However, so far it has been difficult to establish a causal relationship between the appearance of KT-derived MTs and efficient KT capture. It has also been unclear how much contribution KT-derived MTs make to efficient KT capture by spindle-pole MTs. Here we show that a MT-associated protein Stu1^CLASP is a good molecular tool to study the role of KT-derived MTs. Depletion of Stu1 protein abolished both localisation of the microtubule polymerase Stu2^XMAP215 from KTs, and MT/tubulin nucleation at KTs, without affecting KT assembly (i.e. the ability of KTs to interact with spindle-pole MTs) or generation of spindle-pole MTs. Abolishing these KT-derived MTs in Stu1-depleted cells led to a delay in KT capture with an increase in the average capture time. To test whether KT-derived MTs were solely responsible for this delay in KT capture, we developed a mathematical model to recapitulate the roles of KT-derived MTs in Stu1-depleted cells. The model suggested that Stu1-depletion indeed delays KT capture due to a lack of KT-derived MTs. Our results also revealed the extent to which KT-derived MTs contribute to a rapid KT capture by spindle-pole MTs. Furthermore we showed that, after initial KT capture, KT-associated Stu1^CLASP and Stu^2XMAP215 are required to regulate dynamics of their associated spindle-pole MTs. Removal of Stu1^CLASP and Stu^2XMAP215 from KTs lead to defects in KT-dependent switch from MT depolymerisation to polymerisation (rescue). Interfering with KT-dependent MT rescue would compromise the maintenance of the KT–MT interaction. Our study reveals that KT-derived MTs facilitate efficient KT capture by spindle-pole MTs. Stu1^CLASP promotes MT generation at KTs by recruiting a MT polymerase Stu^2XMAP215. Afterwards, Stu^2XMAP215 recruitment by Stu1^CLASP to KTs is also important for MT rescue and sustained KT–MT interaction. Thus, we reveal crucial regulatory mechanisms of KT–MT interaction in early stages of mitosis.

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Farrell, Megan Christine. "Deciphering the Role of Kinetochores and Microtubules During Interphase and Mitosis in Toxoplasma Gondii." Thesis, Boston College, 2014. http://hdl.handle.net/2345/3824.

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Thesis advisor: Marc-Jan Gubbels
The obligate intracellular parasite Toxoplasma gondii exhibits closed mitosis, as chromosome segregation occurs with the confines of the nuclear envelope. Distinct structural changes are absent during mitosis, as the nucleolus is maintained and condensation of chromosomes is largely restricted. Moreover, the centromeres are clustered and remain persistently associated with the centrocone (spindle pole). To elucidate the process of chromosome segregation during mitosis in the parasite, the role of kinetochores and microtubules was examined. Localization studies of the functionally conserved kinetochore proteins TgNuf2 and TgNdc80 revealed that clustered kinetochores colocalize with clustered centromeres at the centrocone throughout the cell cycle. Pharmacological disruption of microtubules resulted in partial loss of clustering, which indicates spindle microtubules are necessary, but not strictly required for this process. Furthermore, the generation of a conditional TgNuf2 knockdown revealed this kinetochore protein is essential for chromosome segregation but dispensable for clustering of centromeres, which remain associated with the centrocone. Moreover, in the absence of TgNuf2 the centrosome behaves normally, but looses its association with the centrocone. Further analysis of this phenotype revealed that the centrocone is devoid of spindle microtubules following depletion of this essential kinetochore protein. Examination of tubulin localization dynamics through parasite development showed that the initiation of spindle microtubules occurs at the basal region of the nucleus prior to centrosome duplication. Furthermore, acetylation of α-tubulin, a posttranslational modification associated with microtubule stability, was confirmed to be specifically associated with stabilization of the spindle microtubules following comigration of the centrocone and centrosome to the apical end of the nucleus. Collectively, these data demonstrate that the persistent association of clustered centromeres with the centrocone is independent of spindle microtubules. These discoveries are contributing unprecedented details to chromosome anchoring and segregation during the cell cycle in this protozoan parasite
Thesis (PhD) — Boston College, 2014
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

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Ramírez, Cota Rosa María. "Dissecting the function of γTuRC subunits in microtubule nucleation and organization." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/398851.

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The depletion of MZT1 in human cells causes severe mitotic spindle defects. Depleted cells lack centrosomal γ-tubulin and arrest in mitosis with a monopolar spindle configuration. Similarly, double deletion mutants of the plant MZT1 orthologs GIP1a and GIP1b are embryonic lethal due to abnormal spindle microtubule distribution and chromosome mis-segregation. Moreover GIP1a and GIP1b were shown to localize to active nucleation sites in the interphase cortical microtubule array. MZT1 function is conserved in fission yeast, where it interacts with GCP3, and is an essential component for the recruitment of the γ-tubulin complex to the spindle pole body, the centrosome equivalent, but not for assembly of the γ-tubulin complex. However, the molecular mechanisms underlying these effects remain unknown. The main goal of this project is to study how MZT1 regulates γTuRC to control MT nucleation and organization. In this work I confirm that in human cells MZT1 is a subunit of the γTuRC and is required for the targeting of the γTuRC to centrosomes and for proper spindle formation. By sucrose gradient fractionation I found that in mammalian cells, MZT1 is not required to assemble the γTuRC. Interestingly, I found that MZT1 is necessary for the interaction of the γTuRC with the targeting factor NEDD1/GCPWD. While in plants and in fission yeast MZT1 interacts with the N-terminal region of GCP3, I found that in mammalian cells MZT1 interacts with a conserve motif at the N-terminal extension of GCP2, GCP3, GCP5 and GCP6. Furthermore, by immunoprecipitation of FLAG-tagged GCPs MZT1 binding motif mutants, I found that the mutants can be integrated into the γTuRC but lost the interaction with GCP-WD and fail to be targeted to the centrosomes To study the role of MZT1 in MT nucleation I performed a MT regrowth experiment with U2OS cells over expressing the CDK5RAP2 nucleation-activating fragment (CDK5RAP2 CM1) and depleted of MZT1. The MT nucleation induced by CDK5RAP2 was lost upon the depletion of MZT1, suggesting that MZT1 in required for the MT nucleation activation mediated by CDK5RAP2. In summary MZT1 is required for all γTuRC-dependent functions including centriole duplication. MZT1 binds to a conserved motif present in the extended N-termini of GCP2, GCP3, GCP5 and GCP6, allowing specific recognition of fully assembled γTuRC. Binding of MZT1 primes γTuRC for interaction with the adapter NEDD1/GCP-WD for targeting γTuRC to centrosomes. In addition, MZT1-dependent priming is required for the CDK5RAP2 CM1 domain to activate γTuRC nucleation activity. Thus, by enabling specific recognition of γTuRC by targeting and activation factors, MZT1 spatially controls microtubule nucleation
En las células humanas la depleción de MZT1 provoca graves defectos del huso mitótico. Las células deplecionadas carecen de γ-tubulina centrosomal y presentan una detención de la mitosis con una configuración monopolar del huso mitótico. Del mismo modo, mutantes de deleción dobles de planta MZT1 con sus ortólogos GIP1a y GIP1b son letales para los embriones debido a la anormal distribución de los microtúbulos del huso mitótico y la mala segregación del cromosoma. Además, GIP1a y GIP1b localizan en sitios de nucleación activos de los microtúbulos corticales. La función de MZT1 se conserva en la levadura de fisión, donde interactúa con GCP3, y es un componente esencial para el reclutamiento del complejo de γ-tubulina en el huso polar del cuerpo apical, el equivalente al centrosoma, pero no para el montaje de la γ-tubulina compleja. Sin embargo, los mecanismos moleculares que subyacen a estos efectos siguen siendo desconocidos. El objetivo principal de este proyecto es estudiar cómo MZT1 regula la actividad del γTuRC en la nucleación y organización de los microtúbulos. En este trabajo encontré que MZT1 es necesaria para todas las funciones γTuRC-dependientes, como la duplicación de centríolos. MZT1 se une a un motivo conservado presente en la N-terminales extendida de GCP2, GCP3, GCP5 y GCP6, lo que permite el reconocimiento específico de γTuRC totalmente ensamblado. La unión de MZT1 al γTuRC “prepara” al complejo para la interacción con el adaptador NEDD1/GCP-WD para la orientación γTuRC a los centrosomas. Además, se requieren esta “preparación” para activar la actividad nucleadora del γTuRC mediada por CDK5RAP2 CM1. Por lo tanto, al permitir el reconocimiento específico de γTuRC por los factores de reclutamiento y los factores de activación, se observa que la MZT1 controla espacialmente la nucleación de microtúbulos.

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Kuhnert, Oliver. "Charakterisierung der neuen centrosomalen Proteine CP148 und CP55 in Dictyostelium discoideum." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2012/5994/.

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Das im Cytosol liegende Dictyostelium Centrosom ist aus einer geschichteten Core-Region aufgebaut, die von einer Mikrotubuli-nukleierenden Corona umgeben ist. Zudem ist es über eine spezifische Verbindung eng an den Kern geknüpft und durch die Kernmembran hindurch mit den geclusterten Centromeren verbunden. Beim G2/M Übergang dissoziiert die Corona vom Centrosom und der Core verdoppelt sich so dass zwei Spindelpole entstehen. CP55 und CP148 wurden in einer Proteom-Analyse des Centrosoms identifiziert. CP148 ist ein neues coiled-coil Protein der centrosomalen Corona. Es zeigt eine zellzyklusabhängige An- und Abwesenheit am Centrosom, die mit der Dissoziation der Corona in der Prophase und ihrer Neubildung in der Telophase korreliert. Während der Telophase erschienen in GFP-CP148 exprimierenden Zellen viele, kleine GFP-CP148-Foci im Cytoplasma, die zum Teil miteinander fusionierten und zum Centrosom wanderten. Daraus resultierte eine hypertrophe Corona in Zellen mit starker GFP-CP148 Überexpression. Ein Knockdown von CP148 durch RNAi führte zu einem Verlust der Corona und einem ungeordneten Interphase Mikrotubuli-Cytoskelett. Die Bildung der mitotischen Spindel und der astralen Mikrotubuli blieb davon unbeeinflusst. Das bedeutet, dass die Mikrotubuli-Nukleationskomplexe während der Interphase und Mitose über verschiedene Wege mit dem Core assoziiert sind. Des Weiteren bewirkte der Knockdown eine Dispersion der Centromere sowie eine veränderte Sun1 Lokalisation in der Kernhülle. Somit spielt CP148 ebenso eine Rolle in der Centrosomen-Centromer-Verbindung. Zusammengefasst ist CP148 ein essentielles Protein für die Bildung und Organisation der Corona, welche wiederum für die Centrosom/Centromer Verbindung benötigt wird. CP55 wurde als Protein der Core-Region identifiziert und verbleibt während des Zellzyklus am Centrosom. Dort besitzt es strukturelle Aufgaben, da die Mehrheit der GFP-CP55 Moleküle in der Interphase keine Mobilität zeigten. Die GFP-CP55 Überexpression führte zur Bildung von überzähligen Centrosomen mit der üblichen Ausstattung an Markerproteinen der Corona und des Cores. CP55 Knockout-Zellen waren durch eine erhöhte Ploidie, eine weniger strukturierte und leicht vergrößerte Corona sowie zusätzliche cytosolische Mikrotubuli-organisierende Zentren charakterisiert. Letztere entstanden in der Telophase und enthielten nur Corona- aber keine Core-Proteine. In CP55 k/o Zellen erfolgte die Rekrutierung des Corona-Organisators CP148 an den Spindelpol bereits in der frühen Metaphase anstatt, wie üblich, erst in der Telophase. Außerdem zeigten die Knockout-Zellen Wachstumsdefekte, deren Grund vermutlich Schwierigkeiten bei der Centrosomenverdopplung in der Prophase durch das Fehlen von CP55 waren. Darüber hinaus konnten die Knockout-Zellen phagozytiertes Material nicht verwerten, obwohl der Vorgang der Phagozytose nicht beeinträchtigt war. Dieser Defekt kann dem im CP55 k/o auftretenden dispergierten Golgi-Apparat zugeschrieben werden.
The Dictyostelium centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates yielding two spindle poles. The two proteins CP148 and CP55 were discovered in a proteomic analysis of Dictyostelium centrosomes. CP148 is a novel coiled-coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage. As CP148, CP55 was also identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed the majority of centrosomal GFP-CP55 is immobile indicating a structural task of CP55 at the centrosome. GFP-CP55 overexpression elicits supernumerary centrosomes containing the usual set of corona and core marker proteins. The CP55 null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their disorganized Golgi apparatus.

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Guieu, Benjamin. "Synthèse de pyrroles polysubstitués par cyclisation à l'or : évaluation de l'activité de 3-arylpyrroles sur les microtubules." Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1S084/document.

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Des composés de type 3-arylpyrroles appelés pyakols ont montré une activité antimitotique sur des cellules tumorales murines, avec en particulier un effet sur les microtubules. Ce type d’activité biologique présentant un intérêt important en cancérologie, le travail présenté dans ce manuscrit est consacré à l’étude de ces hétérocycles. La première partie a pour objectif de développer une stratégie de synthèse permettant d’accéder efficacement au composé chef de file (pyakol I), basée sur la cyclisation d’intermédiaires α-amino-ynols catalysée par des complexes d’or. L’évaluation de l’activité biologique du pyakol I sur le cycle cellulaire et le cytosquelette de diverses lignées tumorales humaines a été réalisée. Les premiers résultats ont révélé une action originale du pyakol I sur le cytosquelette, provoquant une désorganisation du réseau de microtubules et un défaut de positionnement du fuseau mitotique. La séquence réactionnelle a ensuite été validée en l’appliquant pour la réalisation de modulations autour du motif 3-arylpyrrole ainsi que pour l’obtention de molécules marquées. La deuxième partie concerne un travail de méthodologie basée sur la réaction de cyclisation à l’or pour la synthèse de nouveaux pyrroles trifluorométhylés polysubstitués. La stratégie utilisant le trifluoroacétaldéhyde comme substrat de départ permet d’accéder à divers 3-trifluorométhylpyrroles avec de bons rendements, dans des conditions douces
A family of 3-arylpyrroles named pyakols have shown antimitotic properties on murine cell lines, displaying in particular an effect on microtubules. Given the interest of these properties in cancerology, this work is focused on these heterocycles. The objective of the first part was to develop a synthetic strategy based on the gold-catalysed cyclisation of α-amino-ynols intermediates in order to access the lead (Pyakol I). Then, the evaluation of the biological activity of this molecule on the cell cycle and on the cytoskeleton of various human tumoral cell lines was carried out. The first results revealed an original effect on the organization of the microtubules network and the positioning of the mitotic spindle. The developed strategy was then validated by modulating the 3-arylpyrrole moiety on diverse positions, and used for the synthesis of labelled derivatives. The second part of this manuscript focused on the development of a methodology to synthesize new polysubstituted 3-trifluoromethylpyrroles, based on the gold-catalyzed cyclisation reaction. Using trifluoroacetaldehyde as building-block, various trifluoromethylated pyrroles were obtained in mild conditions with good yields

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Argenty, Jérémy. "Rôles dans les lymphocytes T de la protéine Lis1, un régulateur de la dynamique des microtubules dépendante de la dynéine." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30123/document.

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Les récepteurs d'antigènes des lymphocytes T (TCR) sont assemblés au cours du développement précoce de ces cellules dans le thymus suite à des recombinaisons complexes de gènes. Le réarrangement d'une chaine beta des TCR fonctionnelle (pré-TCR) déclenche des voies de signalisation intracellulaires qui entrainent la survie, l'expansion et la maturation des thymocytes. Par ailleurs, l'engagement des TCR à la surface des lymphocytes T (LT) matures par des antigènes conduit également à des cycles de prolifération qui permettent le développement de réponses immunitaires efficaces. Ces évènements cellulaires s'accompagnent de remaniements importants du réseau de microtubules et une redistribution des moteurs moléculaires, tels que la dynéine, qui véhiculent les structures cellulaires sur ces réseaux. Les mécanismes moléculaires et les conséquences physiologiques de ces remaniements sont peu connus dans les LT. Lis1 est un régulateur de la dynéine qui est mis à contribution dans la migration neuronale et la prolifération des cellules souches au cours du développement neural. Son rôle au sein du tissu lymphoïde est peu connu. Dans ce travail, nous avons utilisé des modèles de souris spécifiquement déficients en Lis1 dans les LT afin d'étudier les fonctions moléculaires, cellulaires et physiologiques de cette protéine dans ces cellules. Nous montrons que Lis1 joue un rôle essentiel dans le développement précoce des LT et dans l'homéostasie des LT matures. La déficience en Lis1 n'affecte pas le réarrangement de la chaine beta ou les évènements de signalisation déclenchés par le pré-TCR ou le TCR. Cependant, la prolifération des thymocytes ayant passé la beta-sélection ou des LT matures dont le TCR a été engagé, est fortement impactée. L'analyse fine de la mitose indique que la déficience en Lis1 ralentit fortement le processus mitotique, contrarie les remaniements intracellulaires conduisant à la métaphase et entraîne la répartition asymétrique du matériel génétique dans les cellules filles. L'analyse des réseaux de microtubules montre que l'absence de Lis1 entraîne l'amplification du nombre de centrosomes et l'augmentation des cellules multipolaires au cours de la mitose. Enfin, nous montrons que Lis1 favorise l'interaction de la dynéine avec la dynactine, indiquant que Lis1 joue un rôle important dans les LT pour relier la dynéine aux structures cellulaires qu'elle véhicule. En conclusion, nous avons montré que Lis1 est importante dans la distribution du matériel génétique au cours de la prolifération des thymocytes doubles négatifs et des lymphocytes T périphériques
The T cell receptor (TCR) is assembled during the early development of T lymphocytes in the thymus after complexe genetic recombinations. The rearrangement of a functional TCR beta-chain (pre-TCR) triggers intracellular signaling pathways that cause the survival, expansion and maturation of thymocytes. The commitment of the TCR to the surface of mature T cells after antigen recognition also leads to proliferation allowing the development of effective immune responses. These cellular events go along with significant reorganization of the microtubule networks and a redistribution of molecular motors, such as dynein, which transport the cellular structures via this network. The molecular mechanisms and physiological consequences of the reorganization are poorly understood in T cells. Lis1 is a dynein regulator involved in neuronal migration and stem cells proliferation during neural development. Its role in lymphoid tissue is still unknown. In this study, we used mouse models specifically Lis1-deficient in T cells to study the molecular, cellular and physiological functions of this protein in T cells. We identifiy that Lis1 plays an essential role in the early development of T cells and in the homeostasis of mature cells. Lis1 deficiency does not affect beta-chain rearrangement or signaling events triggered by pre-TCR or TCR, but leads to the blockage of thymocyte cell division that have undergone beta-selection or mature T cells stimulated. Fine analysis of mitosis indicates that the deficiency of Lis1 strongly slows down the mitotic process, counteracts the cell changes leading to the metaphase and leads to asymmetric distribution of the genetic material in the daughter cells. Microtubule networks analysis shows that the absence of Lis1 induces centrosomes amplification and increase of multipolar cells during mitosis. Finally, we show that Lis1 promotes the dynein-dynactin interaction, indicating that Lis1 plays an important role in T cells to bind dynein to the cell structures it carries. In conclusion, we here described that Lis1 is important for the distribution of genetic material during double negative thymocyte and peripheral lymphocyte proliferation

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Masoud, Kinda. "Caractérisation moléculaire et fonctionnelle des protéines GIPs (Gamma-tubulin complex protein 3-Interacting Proteins) d'Arabidopsis thaliana." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ011/document.

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Les microtubules constituent l’un des réseaux du cytosquelette des cellules eucaryotes. Ils jouent un rôle central dans de multiples fonctions comme la division cellulaire, les trafics intracellulaires et la morphogenèse cellulaire. Chez les plantes supérieures, les microtubules (MTs) forment différents réseaux qui s'assemblent au cours du cycle cellulaire. Cette spécificité nécessite un recrutement régulé des complexes de nucléation des MTs à l’enveloppe nucléaire, au cortex et au niveau de MTs préexistants, qui sont des sites de nucléation caractérisés. L'équipe d’A.C. Schmit (IBMP, CNRS, Strasbourg), dans laquelle j'ai effectué mon travail de thèse, se focalise sur la caractérisation des complexes de nucléation des MTs (γ-TuRCs) et la régulation de l'assemblage du fuseau mitotique chez les plantes. Deux nouvelles protéines associées au γ-TuRC ont été mises en évidence par une interaction directe avec l'un de ses composants AtGCP3. Ces protéines, AtGIP1 et AtGIP2 (GCP3 Interacting Protein 1 et 2), sont très conservées au cours de l'évolution, mais leur fonction reste totalement inconnue. Mon travail a été consacré à la caractérisation de cette nouvelle classe de protéines dans le but de comprendre leur rôle. Nos résultats suggèrent que l'association des protéines GIPs aux γ-TuRCs participe à la régulation de leur activité et à la formation d'un fuseau mitotique robuste. Le profil de localisation des protéines GIPs au cours du cycle cellulaire et les phénotypes observés chez les mutants "perte de fonction" gip1gip2 indiquent que ces protéines interviennent dans le recrutement des γ-TuRCs, la nucléation des MTs, l’assemblage du fuseau mitotique, le déroulement du cycle cellulaire et l'organisation des méristèmes. L’étude des mécanismes de régulation de cette famille de protéines a été initiée. Nos résultats ont permis d’identifier GIP1comme un substrat de la kinase Aurora1 in vitro. Les résultats d’expérience de complémentation avec des phosphomutants GIP1 indiquent que la/les fonction(s) des GIPs pourrai(en)t être dépendante(s) de la phosphorylation par la kinase Aurora1, qui est un régulateur avéré du cycle cellulaire. L’ensemble de mes travaux a ainsi contribué à la caractérisation de nouveaux acteurs du cytosquelette microtubulaire. Une meilleure connaissance de leur réseau d'interaction (interactome) ainsi que l’étude de leur homologue humain pourraient ouvrir de nouvelles perspectives de recherche dans le contrôle de la division cellulaire et la lutte contre le cancer
Microtubules (MTs) constitute one of the cytoskeletal networks in eukaryotic cells. They are involved in various processes such as cell division, intracellular transport and cell morphogenesis. In higher plants, MTs can be organized into dynamic structures, which undergo continual assembly and disassembly during the cell cycle. This specificity requires the recruitment of the nucleation complexes of the MTs to the nuclear envelope, to the cortex and to pre-existing MTs. The work of A. C. Schmit’s team (IBMP, CNRS, Strasbourg), in which I did my thesis, focuses on the characterization of MT nucleation complexes (γ-TuRCs) and the regulation of mitotic spindle assembly in plants. We have identified small proteins interacting with Gamma-tubulin Complex Protein 3 (GCP) and named GIP1 and GIP2 (GCP3-Interacting Proteins). The aim of these studies was to characterize this new class of proteins in order to understand their role. It shows that GIPs are conserved among eukaryotes and suggests that their association with the γ-TuRC participates in the regulation of their activity and the formation of a robust mitotic spindle. The localization of GIPs during the cell cycle and the phenotypes observed in T-DNA insertional gip1gip2 double mutants indicatethat GIPs are required for the recruitment of γ-TuRCs, MT nucleation, spindle assembly, cell cycle regulation and stem cell maintenance. Likewise, in vitro assays showed that GIP1 is a novel substrate for Aurora kinase1, which is a well known cell cycle regulator. The results of complementation experiments with GIP1 phosphomutants indicate that the phosphorylation of GIPs may be required for their function(s). Altogether, our results have contributed to the characterization of a new class of proteins involved in MT nucleation/organization and functions. The study of the interaction network (interactome) of GIPs and oftheir homologues could open new ways of research in the control of cell division and in the fight against cancer

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Albertini, Catherine. "Mise en evidence de proteines associees aux microtubules chez physarum polycephalum." Toulouse 3, 1988. http://www.theses.fr/1988TOU30020.

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Nahaboo, Wallis. "Élongation du fuseau mitotique dans l'Embryon de C. elegans : caractérisation d'une Nouvelle Force de propulsion." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEN003.

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A la fin de la vie d’une cellule, différentes forces mécaniques permettent la séparation des chromosomes. Nos données préliminaires suggèrent l’existence d’un autre mécanisme provenant du centre du fuseau mitotique, non décrit dans l’embryon une cellule de C. elegans qui permettrait la séparation des chromosomes. Dans cette cellule, les microtubules kinétochoriens n’appliquent aucune force mécaniques sur les chromosomes durant l’anaphase. Il a été décrit que les chromosomes sont séparés grâce au déplacement des centrosomes via les forces de traction corticales. A l’aide de la microchirurgie laser dans les embryons une cellule de C. elegans, j’ai montré qu’en détruisant physiquement un ou deux centrosomes, les chromosomes continuent de se séparer, révélant l’existence d’une force de propulsion interne au fuseau mitotique (Nahaboo et al., 2015). En combinant la destruction de centrosomes et l’inactivation génétique, nous avons caractérisé les rôles de gènes favorisant ou freinant cette force de propulsion. J’ai observé que la kinésine-5, BMK-1, et le crosslinker MAP-65/SPD-1 freinent cette force de propulsion. Alors que dans d’autres espèces ces protéines favorisent la séparation des chromosomes. Nous avons remarqué que les protéines RanGTP et CLASP, favorisant de la nucléation et la polymérisation des microtubules, aident cette force de propulsion. Ces propriétés suggèrent que la polymérisation des microtubules au centre du fuseau est requise pour permettre la séparation des chromosomes durant la mitose.Par manque d’outils adéquats afin d’altérer la dynamique des microtubules, nous avons collaboré avec l’équipe de biochimistes du Dr. D. Trauner à Munich en Allemagne. Ils ont synthétisé la molécule photoactivable, Photostatin (PST), permettant la dépolymérisation des microtubules en quelques secondes (Borowiak et al., 2015). Entre 390 - 430 nm, PST est activé, dépolymérisant les microtubules, alors qu’entre 500 – 530 nm, PST est inactivé, permettant la polymérisation normale des microtubules. J’ai mesuré que la croissance des microtubules avec PST actif est absente dans des cellules Hela. J’ai montré que le cycle cellulaire dans l’embryon de C. elegans est arrêté localement en présence de PST actif. Nous avons alors montré que PST contrôle optiquement la dynamique des microtubules, in vitro, in cellulo et in vivo, de manière non invasive, rapide, locale et réversible. En résume, j’ai identifié une nouvelle force permettant la séparation des chromosomes à l’aide des approches moléculaires et biophysiques, et j’ai aidé à la caractérisation PST, un antimicrotubule photoactivable de manière locale et réversible
In mitosis, different mechanical forces are involved in chromosome segregation. In C. elegans one-cell embryos, preliminary data suggest that an unknown mechanism, coming from inside the mitotic spindle, could influence chromosome separation. In those cells, it has been showed that kinetochore microtubule activity is absent. Thanks to external pulling forces, centrosome separation drives chromosome segregation. By using microsurgery inside the one-cell C. elegans embryos, we have shown that destroying one or two centrosomes did not prevent chromosome separation, revealing the existence of an outward pushing force (Nahaboo et al., 2015). By combining gene inactivation and centrosome destruction, we showed that the kinesin-5 and the crosslinker SPD-1 act as a brake on this pushing force, whereas they enhance chromosome segregation in other species. Moreover, we identified a novel role for the two microtubule-growth and nucleation agents, RanGTP and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is required to sustain chromosome segregation during mitosis. Then, we aim to reversibility affect microtubule dynamics in the central spindle. Because of the lack of adequate tools, we have collaborated with biochemists from Dr. D. Trauner lab, in Munich, Germany, who are specialized in photoactivable drugs. They have synthetized a photoswitable drug, Photostatin (PST), which can depolymerize microtubules in few seconds in an on/off manner (Borowiak et al., 2015). Under blue light (390 - 430 nm), PST is activated leading to microtubule depolymerization, whereas under green light (500 - 530 nm), PST is activated which does not affect microtubule dynamics. I measured that microtubule growing is absent in presence of activated PST in Hela cells. I also showed that cell cycle can be stopped thank to activated PST in multiple cell C. elegans embryos. We have shown that PST can control microtubule dynamics thanks to visible light in vitro, in cellulo and in vivo, as an on/off switch, in a non-invasive, local and reversible manner

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Paganelli, Laëtitia. "Étude de partenaires protéiques d’une protéine associée aux microtubules, MAP65-3, indispensable à la formation des cellules géantes induites par le nématode à galles Meloidogyne incognita : caractérisation du complexe de surveillance de la mitose chez Arabidopsis." Thesis, Nice, 2013. http://www.theses.fr/2013NICE4028/document.

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Les nématodes à galles du genre Meloidogyne sont des parasites obligatoires des plantes. Lors de l’interaction compatible, ils induisent la formation de cellules nourricières hypertrophiées et plurinucléées leur permettant d’assurer croissance et reproduction. L'étude des mécanismes moléculaires impliqués dans la formation de ces cellules géantes a permis d’identifier une protéine associée aux microtubules, MAP65-3, essentielle à la formation de ces cellules géantes et au développement du nématode. Un des partenaires protéiques de MAP65-3 est un homologue de BUB3, membre du « Mitotic Checkpoint Complex » (MCC). Le MCC est un point de contrôle de la mitose assurant la fidélité de la ségrégation des chromosomes. Au cours de ma thèse, j'ai caractérisé chez la plante modèle Arabidopsis thaliana les homologues du MCC: BUB3.1, MAD2 et la famille multigénique composée de BUBR1, BRK1 et BUB1.2. J’ai démontré les interactions in planta entre les membres du complexe, certaines interactions ayant lieu au niveau des noyaux, voire au niveau des centromères. J’ai réalisé l’analyse fonctionnelle de ces gènes et montré qu’ils étaient exprimés dans les tissus enrichis en cellules en division comme MAP65-3. L’étude de la localisation subcellulaire des protéines a révélé une localisation cytoplasmique pour BUB3.1, BUB1.2 et MAD2, nucléaire pour BUBR1 et centromérique pour BRK1. Nous avons pu également montrer que lorsque des défauts d’attachement des microtubules du fuseau mitotique sont provoqués, BUB3.1, BUBR1 et MAD2 se relocalisent au niveau des kinétochores. L’étude de la famille BUB1/BUBR1 a révélé que l’inactivation des gènes correspondants induisait une sensibilité accrue à un traitement chimique déstabilisant les réseaux de microtubules. L’étude de la mitose chez ces mutants a révélé que BUBR1 est essentielle à la réalisation d’une mitose sans erreur chez Arabidopsis. Ce travail a ainsi permis de caractériser pour la première fois le MCC chez A. thaliana
Root-knot nematodes from the genus Meloidogyne are obligate biotrophic plant parasites. During a compatible interaction, they induce the redifferentiation of root cells into multinucleated and hypertrophied feeding cells to ensure their growth and reproduction. The study of molecular and cellular mechanisms underlying giant cell ontogenesis has led to the identification of a Microtubule-Associated Protein, MAP65-3, essential for giant cell ontogenesis and nematode development. One of the MAP65-3 interacting partners is a BUB3 homologue, member of the Mitotic Checkpoint Complex (MCC). The MCC is a surveillance mechanism ensuring that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the mitotic spindle. During my thesis, I have characterized the Arabidopsis thaliana orthologs of the MCC, BUB3.1, MAD2 and the multigenic family composed of BUBR1, BRK1 et BUB1.2. I have demonstrated that MAP65-3 and all the MCC members interact together in planta, some interactions taking place within the nuclei or at the centromeres. As MAP65-3, all these genes are expressed in dividing cells. The study of the subcellular localization of the protein showed a cytoplasmic localization for BUB3.1, BUB1.2 and MAD2, nuclear for BUBR1 and centromeric for BRK1. Thus, the MCC proteins did not relocalize to the kinetochore during a normal mitosis in planta. BUB3.1, BUBR1 and MAD2 localize to the unattached kinetochores following defects in spindle assembly as observed in cells treated with microtubule poisons. The functional analysis of BUB1/BUBR1 multigenic family showed that the knock-out mutants were more sensitive to microtubule-destabilizing drugs. Furthermore, analysis of mitosis revealed that BUBR1 is essential for an error-free mitosis in Arabidopsis. This work represents the first characterization of the MCC in A. thaliana

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Kim, Haein. "Temporal Coordination Of Mitotic Chromosome Alignment And Segregation: Structural And Functional Studies Of Kif18a." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/930.

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Chromosome alignment is highly conserved in all eukaryotic cell divisions. Microtubule (MT) -based forces generated by the mitotic spindle are integral for proper chromosome alignment and equal chromosome segregation. The kinetochore is a multi-subunit protein complex that assembles on centromeric regions of chromosomes. Kinetochores tether chromosomes to MTs (K fibers) that emanate from opposite poles, in a process called biorientation. This linkage translates K fiber dynamics into chromosome movements during alignment and segregation. Stable, high-affinity kinetochore attachments promote spindle assembly checkpoint (SAC) silencing, which is active when unattached kinetochores are present. During chromosome alignment, 1) K fiber plus-end dynamics decrease, confining chromosome movements near the spindle equator, and 2) electrostatic interactions between kinetochore proteins and MTs increase. Chromosome segregation occurs as soon as all chromosomes are stably attached to microtubules and the SAC has been silenced. SAC silencing and chromosome alignment are temporally coordinated during normal divisions, implying that the mechanisms regulating K fiber dynamics and kinetochore affinity must be linked. Interestingly, HeLa cells depleted of a kinesin-8 motor Kif18A, known for its role in promoting chromosome alignment, display a SAC-dependent mitotic delay due to kinetochore-MT attachment defects. This is puzzling, as Kif18A's function in chromosome alignment is to suppress MT growth by stably associating with MT plus-ends. Whether Kif18A is required for attachment in all cells and how it promotes kinetochore microtubule linkages are not understood. The work presented in this dissertation supports a model in which Kif18A functions as a molecular link that coordinates chromosome alignment and anaphase onset. We find that Kif18A is required to stabilize kinetochore-MT attachments during mammalian germline development, as germline precursor cells in Kif18A mutant mice are unable to divide during embryogenesis due to an active SAC. However, while all cell types require functional Kif18A for chromosome alignment, mouse primary somatic cells can still divide with normal timing. This finding indicates a cell-type specific dependence on Kif18A for stabilizing kinetochore-MT attachments, and provides evidence that this function might be separate from Kif18A's known role in chromosome alignment. Consistent with this idea, we find that an evolutionarily conserved binding motif for protein phosphatase 1 (PP1) is required for Kif18A's novel role in regulating kinetochore microtubule attachments. Kif18A-PP1 interaction is required for Kif18A-mediated dephosphorylation of the kinetochore protein Hec1, which enhances attachment. However, Kif18A's interaction with PP1 is dispensable for chromosome alignment. Thus, point mutations that disrupt PP1 binding separate Kif18A's role in stabilizing kinetochore attachments from its function in promoting chromosome alignment. Additionally, through structure function studies of the motor domain, we identified a long surface loop (Loop2) that is required for Kif18A's unique MT plus-end binding activity, which is essential for its function in confining chromosome movements. Taken together, we find that Kif18A is molecularly tuned to provide temporal control of chromosome alignment and anaphase entry.

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Maillet, Vanessa. "LKB1, gardien de la prolifération hépatocytaire et de l’intégrité génomique." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB103/document.

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La Liver Kinase B1 (LKB1) est une protéine pléiotrope, impliquée dans divers processus biologiques. Dans le foie, LKB1 est notamment connue pour être un régulateur clé du métabolisme et de la polarité cellulaire. Au cours de notre étude, nous avons investigué l’implication de LKB1 dans le contrôle de la prolifération des hépatocytes au cours du processus de régénération hépatique physiologique (hépatectomie partielle des 2/3). Nous avons démontré que la perte de Lkb1, spécifiquement dans les hépatocytes, favorise la récupération de la masse hépatique après hépatectomie partielle, en induisant une augmentation drastique de la réponse proliférative hépatocytaire, indépendamment de la balance métabolique/énergétique. Ainsi, LKB1 agit comme un senseur négatif de la prolifération et régule la transition G0/G1, en particulier en contrôlant la signalisation de l’EGFR (Epidermal Growth Factor Receptor). Par ailleurs, plus tard pendant la régénération, LKB1 garantit également l’intégrité mitotique. En effet, la suppression de Lkb1 entraîne des altérations majeures de la formation du fuseau mitotique. Nos résultats établissent également que LKB1 contrôle la polarité de la division cellulaire, indépendamment de l'activité de l’AMPK (AMP-activated protein kinase), une cible clé de LKB1. Par conséquent, la perte de LKB1 conduit à une altération majeure du profil de ploïdie, au stade tardif du processus de régénération. L’ensemble de notre étude souligne le double rôle de LKB1, au cours de la régénération hépatique, en tant que gardien de la prolifération hépatocytaire et de l'intégrité génomique
Liver Kinase B1 (LKB1) is involved in pleiotropic biological processes and known to be a key regulator of hepatic metabolism and polarity. Here, we investigated the contribution of LKB1 in hepatocyte proliferation and liver regeneration process. We demonstrated that loss of hepatic Lkb1 promotes liver mass recovery, through an increase of hepatocytes proliferation, independently on metabolic/energetic balance. LKB1 regulates G0/G1 progression, specifically by controlling Epidermal Growth Factor Receptor (EGFR) signaling. In addition, later during regeneration, LKB1 controls mitotic fidelity. Deletion of Lkb1 results in major alterations of mitotic spindle formation, along the polarity axis, independently of AMP- activated protein kinase (AMPK) activity, a key target of LKB1. Consequently, LKB1 deficiency leads to an alteration of ploidy profile, at late stage of regenerative process. Overall our study highlights the dual role of LKB1, during liver regeneration, as a guardian of hepatocyte proliferation and genomic integrity

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Sdelci, Sara. "Role of the Kinases NEK6, NEK7 and NEK9 in the Regulation of the Centrosome Cycle." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/96820.

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This thesis project is focused on the study of the signaling module formed by the NIMA-related protein Nek6, Nek7, and Nek9 and their function during early mitosis, with particular interest in centrosome separation and maturation. Nek9/Nercc1 was identified by Dr. Joan Roig. Nek9 is expressed in all cell lines and tissues studied is inactive during interphase while during mitosis is activated through phosphorylation by Plk1 which is in fact able to bind Nek9 and subsequently phosphorylates Nek9 on its activation loop. During mitosis Nek6 and Nek7 bind the C-terminal of Nek9. Once active, Nek9 can phosphorylate Nek6 and Nek7, thus activating them. Active Nek9 localizes at centrosome, suggesting that Nek9/Nek6-7 has important functions in the organization of microtubules during cell division. Confirming this idea, it has been shown that the microinjection of anti-Nek9 module induces arrest in prometaphase with disorganized spindle structures and misaligned chromosomes, or leads to abnormal mitosis resulting in aneuploidy. In the same direction, interference with the function of Nek7 or Nek6 leads to abnormal mitotic progression and spindle formation. We described how the Nek9/Nek6-7 module could provide a link connecting Plk1 and Eg5 in the context of centrosome separation. we analyzed the effects of Plk1, Eg5, Nek9, Nek6 or Nek7 down-regulation by RNAi on the extent of separation of duplicated centrosomes in prophase cells and we observed how this downregulation was affecting centrosome separation. We determine whether the activation of Nek9 or Nek6 could induce centrosome separation trasfecting cells with the active form of these two kinases; a considerable amount of cells that were in interphase shown separate centrosome demonstrating that Nek9/Nek6 are sufficient to induce centrosome separation. To test whether active Nek9 and Nek6 exerted their effect through the regulation of Eg5 we simultaneously transfected the cells with Eg5 siRNAs and we completely lost the centrosome separation described above. We demonstrated by immunofluorescence that the key event during centrosome separation was the recruitment of Eg5 at centrosomes and that the down-regulation of Plk1, Nek6, Nek7 or Nek9 resulted in prophase cells with unseparated centrosomes because Eg5 was not properly recruited. To prove whether the phosphorylation on Ser-1033 controls the accumulation of Eg5 to centrosomes and centrosome separation during early mitosis we transfected cells with wild type Eg5 or Eg5 S1033A; the wild type form of the kinesin was able to localize at centrosome and rescue the normal phenotype while Eg5 S1033A was not able to localize and resulted in cells delayed in mitosis. Plk1, the Nek9 activator, is involved in the regulation of centrosome maturation during early mitosis. Centrosome maturation refers to the process through which centrosomes increase size and microtubule nucleation activity and requires the accumulation of γ-TuRC complexes at centrosome. This recruitment depends on Nedd1 that acts as γ-Tubulin targeting factor. Plk1 depletion prevents accumulation of Nedd1 at centrosome. Our experiments show the importance of Nek9 in the regulation of centrosome maturation downstream of Plk1. Depletion of Nek9 by siRNA determined a decrease of γ-Tubulin and Nedd1 at centrosome. Further we investigated the upstream role of Plk1 depleting Plk1 and trasfecting active Nek9 and it was able to rescue the normal phenotype. Nek9 can interact with Nedd1 during mitosis and phosphorylates it provoking its accumulation at centrosome. The no-phosphorylable form of Nedd1 was not able to accumulate at centrosome and support the accumulation of γ-Tubulin there, determining a delay of the cells in prometaphase. Our results show that Nek9 is the link between Plk1 activity and the recruitment of Nedd1 to the centrosome and that the pathway formed by Plk1/Nek9/Nedd1 can be a key element in the control of mitotic centrosome maturation.

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Müller-Reichert, Thomas. "Spindle organization in three dimensions." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1166107130476-22269.

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During cell division, chromosome segregation takes place on bipolar, microtubulebased spindles. Here, C. elegans is used to analyze spindle organization under both mitotic and meiotic conditions. First, the role of SAS-4 in organizing centrosome structure was analyzed. Partial depletion of SAS-4 in early embryos results in structurally defective centrioles. The study of this protein sheds light on the poorly understood role of the centrioles in dictating centrosome size. Second, the ultrastructure of wild-type mitotic spindle components was analyzed by electron tomography. This 3-D analysis reveals morphologically distinct microtubule end morphologies in the mitotic spindle pole. These results have structural implications for models of microtubule interactions with centrosomes Third, spindle assembly was studied in female meiosis. Specifically, the role of the microtubule severing complex katanin in spindle organization was analyzed. Electron tomography reveals fragmentation of spindle microtubules and suggests a novel katanin-dependent mechanism of meiotic spindle assembly. In this model, relatively long microtubules seen near the meiotic chromatin are converted into numerous short fragments, thus increasing the total number of polymers in an acentrosomal environment. Taken together, these results provide novel insights into the three-dimensional organization of microtubules during spindle assembly
Die Segregation der Chromosomen während der Zellteilung wird duch bipolare, von Microtubuli-aufgebauten Spindlen gewährleistet. In der vorliegenden Arbeit wird C. elegans zur Analyse der Spindelorganisation unter mitotischen und meiotischen Bedingungen herangezogen. Erstens wird die Rolle von SAS-4 in der Organisation von Zentrosomen untersucht. Die partielle Depletierung von SAS-4 in frühen Embryonen führt zu strukturell defekten Zentriolen und wirft somit Licht auf die wenig verstandene Rolle der Zentriolen in der Bestimmung der Zentrosomengröße. Zweitens wird die Ultrastruktur der mitotischen Spindelkomponenten im Wildtyp durch Elektronentomographie untersucht. Diese 3-D-Analyse zeigt, dass im mitotischen Spindlepol unterschiedliche Morphologien der Mikrotubulienden zu finden sind. Diese Ergebnisse haben strukturelle Implikationen für Modelle der Mikrotubuli-Zentrosomen-Interaktionen. Drittens wird der Aufbau der Spindel in der weiblichen Meiose, speziell die Rolle des Mikrotubuli-schneidenden Kataninkomplexes in der Spindelorganisation, untersucht. Die Elektronentomographie zeigt hier eine Fragmentierung der Spindelmikrotubuli. Basierend auf diesem Ergebnis wird ein neues Katanin-abhängiges Modell der Formierung der Meiosespindel entwickelt, in dem relativ lange Microtubuli in Nähe des meiotischen Chromatins in zahlreiche kurze Mikrotubuli “zerschnitten” werden. Dies erhöht die Anzahl der verfügbaren Polymere in dieser azentrosomalen Situation. Zusammenfassend bringen diese Ergebnisse neue Einsichten in die räumliche Organisation der Mikrotubuli während des Spindelaufbaus

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18

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-MTs
The 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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19

Hunter, Andrew W. "Coupling of ATP hydrolysis to microtubule depolymerization by mitotic centromere-associated kinesin /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10549.

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20

Ferreira, Vanessa Miriam dos Reis. "The role of phosphorylation in the regulation of the chromokinesin Xkid." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/31906.

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Xkid is a Xenopus chromokinesin required for metaphase chromosome alignment and for meiosis I to meiosis II transition in oocytes. The aim of this work was to study the regulation of Xkid by phosphorylation using the Xenopus oocyte and egg extract systems. To achieve this, a reliable method to express proteins in egg extract by addition of in vitro transcribed mRNAs was established. Xkid was found to be efficiently phosphorylated in meiosis and mitosis at the cdk1 site. Although phosphoXkid localized efficiently to the mitotic chromosomes, phosphorylation at the cdk1 site had no role in the binding of Xkid to the chromosomes but prevented the protein to localize to the spindle microtubules like the endogenous protein. The dominant negative effect on spindle assembly of a phospho mimicking form of Xkid indicated that phosphorylation plays an important role in the regulation of Xkid function. Several partners for Xkid were identified.
Xkid es una cromoquinesina del sistema de Xenopus, necesaria para el alineamiento de los cromosomas en la placa metafásica y para la transición entre la meiosis I y meiosis II en los oócitos. El objectivo de este trabajo era estudiar la regulación de Xkid por fosforilación en los oócitos y en el extracto de huevos de Xenopus. Para poder cumplirlo se estableció un método para la expresión de proteínas añadiendo al extracto de huevos ARN mensajeros sintetizados in vitro. Los resultados obtenidos sugieren que Xkid es eficientemente fosforilada en el sitio cdk1 durante la meiosis y la mitosis. Aunque la forma de Xkid fosforilada se localiza eficientemente a nivel de los cromosomas mitóticos, esta fosforilación no parece tener ningún papel regulador sobre esta localización. En cambio, parece interferir con la localización de Xkid sobre los microtúbulos mitóticos. El efecto dominante negativo de la forma de Xkid que mimetiza la fosforilación durante la formación del huso mitótico, sugiere además que la fosforilación desempeña un papel importante en la regulación de la función de Xkid. Finalmente, varias proteínas que interaccionan con Xkid han sido identificadas.

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21

Rosas, Salvans Miquel 1987. "Understanding RanGTP dependent microtubule assembly : Idenification of DnaJB6 as a RanGTP regulated factor involved in microtubule organization during mitosis." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/664169.

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Three microtubule (MT) assembly pathways participate in the assembly of the bipolar spindle: the centrosomal pathway, the augmin dependent amplification pathway and the RanGTP/chromosome dependent pathway. To form the spindle, all these MTs are organized by various classes of motor proteins into two interdigitating antiparallel arrays with their minus ends focused at the spindle poles. This focusing activity is provided by the minus-end directed motor proteins Dynein-Dynactin and HSET. Spindle assembly can occur in the absence of centrosomes indicating that the RanGTP and augmin dependent pathways are sufficient. The RanGTP pathway can be studied in Xenopus laevis egg extracts. Addition of RanGTP to these extracts triggers a dynamic process of MT nucleation, stabilization and organization into asters and mini spindles. To obtain a global picture of the RanGTP pathway we used a proteomics approach and determined the interactome of the RanGTP-MTs that consists of 1263 proteins. Moreover we have analyzed the changes in this proteome to try to correlate them with the change in MT dynamic and organization observed upon different time of incubation of the egg extract with RanGTP. Although the composition of the proteome did not change, we found different patterns of recruitment for various protein groups. The proteome includes most of the known RanGTP regulated factors in mitosis and significantly overlaps with previously published spindle and taxol-MT proteomes. In addition it contains a large number of other proteins with described or undescribed functions in various cellular processes. We used this proteome to identify novel putative RanGTP regulated spindle assembly factors (SAFs). We identified DnaJB6 as a RanGTP regulated protein involved in spindle assembly. We found that it interacts with dynactin p150 in a RanGTP dependent manner specifically in M-phase. We show that DnaJB6 favors the stabilization of the Dynactin complex specifically in mitosis, regulating the activity of Dynein-Dynactin complex in bipolar spindle assembly and MT focusing at the spindle poles.
Tres vies de formació de microtúbuls (MT) participen en la formació del fus mitòtic: la centrosòmica, la via d’amplificació dependent d’Augmin i la via dependent de RanGTP o cromosòmica. Per formar el fus, tots aquests MTs són organitzats per diferents classes de proteïnes motores en dos feixos interconnectats de MTs antiparal·lels, amb els seus extrems negatius concentrats al pols del fus. Dynein-Dynactin i HSET s’encarreguen de concentrar els extrems negatius als pols. El fus es pot formar també en absència de centrosomes, indicant que les vies de RanGTP i d’Augmin són suficients per formar-lo. La via de RanGTP es pot estudiar utilitzant extractes d’ous (EE) de Xenopus Laevis. L’addició de RanGTP activa un procés dinàmics de nucleació, estabilització i organització del MTs en asters i mini-fusos. Hem utilitzat la proteòmica com una aproximació per obtenir una visió global de la ruta de RanGTP i em descrit un interactoma dels RanGTP-MTs de 1263 proteïnes. A més, hem analitzat els canvis en aquest proteoma intentant correlacionar-los amb canvis en la dinàmica i l’organització observades al llarg de diferents temps d’incubació de l’EE amb RanGTP. Tot i que la composició del proteoma no varia, hem trobat diferents patrons de reclutament per varis grups de proteïnes. El proteoma inclou la majoria dels factors regulats per RanGTP en mitosis que es coneixen i te un elevat grau de solapament amb altres proteomes del fus i dels Taxol-MTs publicats prèviament. A més, conté un elevat nombre de proteïnes amb i sense roles descrits en varis processos cel·lulars. Hem utilitzat el proteoma dels RanGTP-MTs per identificar nous possibles factors regulats per Ran involucrats en la formació del fus. Hem identificat DnaJB6 com una proteïna regulada per Ran amb una funció en la formació del fus mitòtic. Hem descrit la interacció de DnaJB6 amb p150, dependent de RanGTP específicament en fase M. DnaJB6 afavoreix l’estabilització el complex Dynactin específicament en mitosis, regulant l’activitat de Dynein-Dynactin en l’establiment de la bipolaritat del fus mitòtic i la concentració dels extrems (-) dels MTs als pols del fus mitòtic.

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22

Bouissou, Anaïs. "Rôle de la tubuline gamma et des protéines associées dans la dynamique des microtubules." Toulouse 3, 2011. http://thesesups.ups-tlse.fr/1151/.

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Les microtubules sont des polymères dynamiques, essentiels à la division cellulaire. Ils sont souvent organisés à partir du centrosome où se localise la tubuline Gamma. Cette protéine joue un rôle important dans la nucléation des microtubules. Elle est présente sous forme de deux complexes: un petit complexe (Gamma-TuSC) essentiel à la viabilité et à l'assemblage d'un fuseau bipolaire fonctionnel et un complexe d'organisation supérieure (Gamma-TuRC) nécessaire au déroulement optimal de la mitose. L'objectif de ma thèse a été de caractériser le rôle des protéines spécifiques du Gamma-TuRC. La stratégie a consisté à inhiber ces protéines « non essentielles » par RNAi dans les cellules de drosophile en culture et à analyser les conséquences sur l'organisation et la dynamique du cytosquelette. En interphase, et pour la première fois chez les métazoaires, j’ai mis en évidence un rôle du Gamma-TuRC dans la dynamique des microtubules. Localisé le long des microtubules, ce complexe agit comme un facteur stabilisateur. En mitose, le Gamma-TuRC est présent sur tous les types de microtubules y compris sur les microtubules astraux. La déplétion du Gamma-TuRC induit des défauts de positionnement du fuseau. Ces anomalies sont corrélées à une modification des propriétés dynamiques des microtubules astraux qui participent à la liaison fuseau/cortex. L'ensemble de mes résultats démontre que le Gamma-TuRC participe à la régulation de la dynamique des microtubules en interphase et en mitose. Ce rôle permettrait au complexe d'être impliqué dans des fonctions non-centrosomales, comme l'organisation de sous-réseaux de microtubules ou le positionnement du fuseau
Microtubules are highly dynamic polymers, essential in cell division. They are often organized from the centrosome where the protein Gamma-tubulin plays an important role in microtubule nucleation. Gamma-tubulin acts within two main complexes: a small Gamma-tubulin complex (Gamma-TuSC) is essential for viability and assembly of a functional spindle, and a larger complex (Gamma-TuRC) is required for efficient mitotic progression. The role of Gamma-TuRC-specific proteins is not well defined. Using RNAi-mediated depletion in Drosophila S2 cells, I studied the function of these non-essential Gamma-TuRC proteins in microtubule organisation and dynamics. In interphase, I show for the first time that Gamma-TuRCs, localized along microtubules, regulate microtubule dynamics, acting as pause factors. In mitosis, Gamma-TuRCs are associated with all microtubule subsets, including astral microtubules. The loss of Gamma-TuRCs alters astral microtubule dynamics, correlated with spindle positioning defects. Together, these results demonstrate that Gamma-TuRCs regulate microtubule dynamics in interphase and in mitosis. We propose that Gamma-TuRCs are essential to mediate non-centrosomal functions such as organization of cell type-specific microtubule networks or spindle positioning

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23

Loncar, Ana. "Comparaison de la dynamique du fuseau mitotique et méiotique chez la levure à fission." Thesis, Université Paris sciences et lettres, 2020. https://tel.archives-ouvertes.fr/tel-03174872.

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La division cellulaire est un processus universel chez tous les êtres vivants où les chromosomes dupliqués sont séparés aux pôles cellulaires opposés. La mitose est un type de division cellulaire qui sert à la prolifération des cellules, tandis que la méiose produit des cellules sexuelles, qui sont utilisées dans la reproduction sexuelle d'un organisme. Dans les deux cas, une machine à base de microtubules appelée le fuseau sépare parfaitement les chromosomes. Une séparation précise des chromosomes est primordiale, car les erreurs de ségrégation des chromosomes dans peuvent entraîner une aneuploïdie qui pourrait provoquer des malformations congénitales, le cancer ou la mort cellulaire.La mitose et la méiose font l'objet de recherches depuis de nombreuses décennies, et une pléthore d'acteurs clés a été identifiée et étudiée. Cependant, aucune étude n'a été réalisée sur la comparaison de la dynamique des fuseaux mitotiques et méiotiques dans le même organisme. Dans cette étude, la dynamique du fuseau mitotique et méiotique a été caractérisée et comparée simultanément dans la levure à fission. La comparaison de la dynamique des fuseaux a permis de déterminer qu'il existe trois types de fuseaux distincts - le fuseau mitotique, méiotique I et méiotique II, avec des caractéristiques distinctives. Un mutant de levure à fission déficient en kinésine-5 Cut7 et en kinésine-14 Pkl1 a été utilisé comme outil pour identifier la source des différences dans la dynamique du fuseau mitotique et méiotique. Bien que les fuseaux mitotiques cut7Δpkl1Δ soient bipolaires et capables de séparer les chromosomes, nous montrons que les fuseaux de la méiose I ne parviennent pas à établir la bipolarité et à séparer les chromosomes, ce qui entraîne la formation de zygotes formant moins de quatre spores typiques. Ensuite, nous révélons que la concentration de Pkl1 est réduite en fuseaux méiotiques I par rapport aux fuseaux mitotiques, et identifions la kinésine-14 Klp2 comme la molécule qui coopère avec Pkl1 en antagonisant Cut7 dans la méiose I. De plus, nous avons constaté que la suppression de la dynamique des microtubules dans cut7Δpkl1Δ zygotes restaure bipolarité du fuseau, faisant valoir que les microtubules sont plus dynamiques dans les fuseaux de la méiose I que dans les fuseaux mitotiques.En résumé, ce travail montre que les fuseaux mitotiques et méiotiques sont intrinsèquement différents, et leurs différences proviennent de la kinésine-14 et de la régulation de la dynamique des microtubules
Cell division is a universal process in all living beings where duplicated chromosomes are separated to the opposite cell poles. Mitosis is a cell division type that serves for proliferation of cells, while meiosis produces sex cells, which are used in the sexual reproduction of an organism. In both cases, a microtubule-based machine called a spindle flawlessly separates the chromosomes. Precise chromosome separation is paramount, as any errors in chromosome segregation can result in aneuploidy that may cause congenital defects, cancer or cell death.Mitosis and meiosis have been the focus of research for many decades, and a plethora of key players has been identified and studied. However, no study has been done on comparison of mitotic and meiotic spindle dynamics in the same organism. In this study, mitotic and meiotic spindle dynamics have been characterized and compared simultaneously in fission yeast. Spindle dynamics comparison ascertained that there are three distinct spindle types – mitotic, meiotic I and meiotic II spindles, with distinguishing features. A fission yeast mutant deficient for kinesin-5 Cut7 and kinesin-14 Pkl1 was used as a tool to identify the source of the differences in mitotic and meiotic spindle dynamics. Although cut7Δpkl1Δ mitotic spindles are bipolar and capable of segregating the chromosomes, we show that meiosis I spindles fail to establish bipolarity and separate the chromosomes, resulting in zygotes forming less than typical four spores. Next, we reveal Pkl1 concentration is reduced in meiotic I compared to mitotic spindles, and identify kinesin-14 Klp2 as the molecule that co-operates with Pkl1 in antagonizing Cut7 in meiosis I. Furthermore, we found that suppressing microtubule dynamics in cut7Δpkl1Δ zygotes restores spindle bipolarity, arguing that microtubules are more dynamic in meiosis I spindles than in mitotic spindles.In summary, this work shows mitotic and meiotic spindles are inherently different, and their differences stem from kinesin-14s and microtubule dynamics regulation

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24

Chakraborty, Papia. "Regulation of Nucleoporins in Mitosis." Scholarly Repository, 2007. http://scholarlyrepository.miami.edu/oa_dissertations/54.

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Nucleoporins mediate nucleocytoplasmic trafficking in interphase. In mitosis, upon nuclear envelope breakdown, the role and regulation of Nups remain to be elucidated. An important subcomplex of nucleoporins is the Nup107-160 complex, which, in mitosis, is involved in spindle assembly and nuclear pore re-assembly. Here we show that the level of a key constituent of the Nup107-160 complex- Nup96 is cell cycle regulated. We found that the mechanism involved in regulating Nup96 levels in mitosis is proteolysis by the anaphase-promoting complex (APC). Nup96 interacts with the APC, and its proteolysis can be regulated by Cdc20 and Cdh1. Like the Nup107-160 complex, the APC is localized at kinetochores, centrosomes, and spindles. Disruption of Nup96 levels led to an acceleration of prophase to prometaphase transition and, most importantly, resulted in a delay of G1 progression. Thus, regulation of Nup96 proteolysis in mitosis sets the stage for proper G1 progression. Additionally, we have observed differential regulation of members of the Nup107-160 complex during mitosis and have identified interacting partners of Nup96 at the centrosome which reveal a novel role of nucleoporins in regulating microtubule nucleation.

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25

Scrofani, Jacopo 1984. "Mechanism of RanGTP dependent microtubule assembly during mitosis." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/289621.

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During mitosis, spindle assembly involves different sources of microtubules including centrosomes and chromosomes. While the role of centrosomes has been extensively studied, we still do not fully understand how chromosomes trigger microtubule assembly thereby contributing to the formation of the mitotic spindle. The chromosomal pathway is largely determined by a RanGTP gradient centered on the chromosomes that induces the local activation of spindle assembly factors. To get a better understanding on the RanGTP-dependent microtubule assembly during mitosis we aimed at: i) Identifying new RanGTP regulated proteins involved in spindle assembly. Our results pointed to three novel proteins with a putative mitotic role in the RanGTP pathway. ii) Understanding how the RanGTP pathway regulates microtubule nucleation during mitosis. We found that TPX2 together with Aurora-A and RHAMM are part of a RanGTP-dependent complex that binds and strongly stimulates the -TuRC nucleation activity. iii) Investigating the contribution of the RanGTP pathway to spindle assembly. Our data provided novel evidences that MTs nucleated close to the chromosomes participate in the k-fibers assembly process.
Durante la mitosis, el proceso de ensamblaje del huso mitótico implica diferentes fuentes de microtúbulos incluyendo centrosomas y cromosomas. Mientras que el rol de los centrosomas ha sido extensamente estudiado, no se entiende en su totalidad como los cromosomas inducen la formación de microtúbulos contribuyendo de esta manera al ensamblaje del huso mitótico. La vía de los cromosomas está mayormente determinada por un gradiente de RanGTP, centrado en los cromosomas, que induce la activación local de factores mitóticos. Para entender el mecanismo que promueve el ensamblaje de los microtúbulos vía RanGTP durante la mitosis, este trabajo tuvo los siguientes objetivos: i) La identificación de nuevas proteínas reguladas por RanGTP que participan en el ensamblaje del huso. Nuestros resultados apuntan a tres nuevas proteínas con un posible papel mitótico en la vía RanGTP de ensamblaje de los microtúbulos. ii) El estudio de el mecanismo para el cual RanGTP regula la nucleación de microtúbulos durante la mitosis. Hemos descubierto que TPX2 junto con Aurora-A y RHAMM son parte de un complejo RanGTP dependiente que estimula la actividad de nucleación de el TuRC. iii) El estudio del papel de la vía de RanGTP en el ensamblaje del huso. En particular, nuestros datos proporcionan nuevas evidencias sobre la participación de los MTs nucleados alrededor de los cromosomas en el proceso del ensamble de fibras cinetocóricas.

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Lecland, Nicolas 1984. "Non-centrosomal microtubule nucleation and organization in mitosis." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/299799.

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During mitotic spindle assembly, γ-tubulin ring complexes (γTuRCs) nucleate microtubules at the centrosome, around mitotic chromatin and, by augmindependent recruitment, from pre-existing microtubules. The analysis of these distinct pathways in somatic cells is challenging due to the predominance of centrosomal nucleation. It is also unknown how microtubules derived from different nucleation pathways are organized into the bipolar spindle structure. Minus ends were shown to be present throughout the spindle with a higher concentration near the poles. However, the analysis of minus end dynamics has been prevented by lack of a suitable probe. I have identified the γ-tubulin ring complex (γTuRC) as a reliable marker for noncentrosomal microtubule minus ends in the spindle and have confirmed the accumulation of minus ends in the pole-proximal region. Using cells stably expressing γ-tubulin fused to photoactivatable GFP and mutants of γTuRC subunits, I have demonstrated that the γTuRC is recruited preferentially in the poledistal spindle region, where it associates with microtubule minus ends and then moves poleward along the mitotic spindle. Poleward transport of γTuRC at minus ends depends on the molecular motors dynein, KIFC1 and KIF11. I also discovered that some of the γTuRC that reaches the poles is stably incorporated at the centrosomes, complementing the microtubule-independent centrosome targeting previously described. Using laser ablation of centrosomes, I studied non-centrosomal spindle assembly. At mitotic entry, in the absence of centrosomes, these cells could nucleate microtubules from the nuclear area. These microtubules formed multipolar spindles but cells eventually divided into two daughter cells. However, cells derived from these abnormal mitoses were typically not viable. In summary, by revealing the dynamics of the minus ends of non-centrosomal microtubules, I have provided novel insight into assembly and architecture of the mitotic spindle. In addition, I have shown that centrosomes, even though not essential for somatic cell division, play an important role in the fidelity of spindle assembly and function.
Durante la formación del huso mitótico, los complejos anulares de γ-tubulina (γTuRCs, del ingles γ-tubulin ring complexes) nuclean microtúbulos alrededor de la cromatina mitótica y, mediante la interacción con el complejo de la Augmina, a partir de otros microtúbulos ya existentes. El estudio de estos mecanismos en células somáticas es complejo, debido a la predominancia de la nucleación centrosomal de los microtúbulos en estas células. En la actualidad, aun no se sabe como microtúbulos procedentes de distintas vías de nucleación se organizan en la estructura bipolar del huso mitótico. Se ha observado que los extremos (-) están presentes a lo largo del huso, detectándose una mayor concentración cerca de los polos. A pesar de esto, no se conocen estudios detallados acerca de la dinámica del extremo (-) debido a la ausencia de marcadores adecuados. He observado que el complejo anular de γ-tubulina (γTuRC) es un marcador fiable de los extremos (-) en los microtúbulos acentrosomales presentes en el huso mitótico. Además, he confirmado la acumulación de estos extremos (-) en la región próxima a los polos del huso. Utilizando líneas celulares que expresan de forma estable γ-tubulina fusionada a un GFP foto-activable, y que además han sido transfectadas con isoformas mutantes de subunidades del γTuRC, he demostrado que el γTuRC se recluta preferentemente en regiones del huso alejadas de los polos. Allí el γTuRC se asocia con los extremos (-) de los microtúbulos, para ser transportado a lo largo del huso mitótico en dirección a los polos. El transporte del γTuRC presente en los extremos (-) en dirección a los polos lo realizan los motores moleculares Dineina, KIFC1 y KIF11. También he descubierto que una parte de las moléculas de γTuRC que alcanzan los polos del huso se integran de forma estable en los centrosomas, complementando la vía de incorporación al centrosoma independiente de microtúbulos que ha sido previamente descrita. Usando la técnica de ablación de centrosomas con laser, he estudiado la formación acentrosomal del huso. Cuando estas células entran en mitosis en ausencia de centrosomas, pueden nuclear microtúbulos desde el área nuclear. Estos microtúbulos forman husos multipolares, aunque las células finalmente se dividen en dos células hijas. Las células procedentes de estas mitosis anormales no suelen ser viables. En conclusión, mediante el revelado la dinámica de los extremos (-) de los microtúbulos no centrosomales, he proporcionado nueva información acerca del ensamblaje y arquitectura del huso mitótico. Además, he demostrado que los centrosomas, aunque no son esenciales para la división celular somática, juegan un papel importante en el correcto ensamblaje y función del huso mitótico.

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Demir, Özlem. "Functional Characterization of Microtubule Associated Proteins in ES Cell Division and Neuronal Differentiation." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-163103.

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Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation.

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28

Kondratick, Christine M. "Mitosis related phosphorylation of the neuronal microtubule-associated protein tau /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487949150071694.

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29

Deavours, Bettina Edith. "Microtubule interactions and regulation of the mitotic kinesin-like protein-1 and kinesin-like calmodulin-binding protein." Diss., Virginia Tech, 2001. http://hdl.handle.net/10919/29951.

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Microtubules are essential for many dynamic processes occurring within eukaryotic cells including organelle and vesicular trafficking, motility of cilia and flagella, and mitosis. Microtubules operate in conjunction with the kinesin superfamily of microtubule-dependent motor proteins, which use the energy from ATP hydrolysis to "walk" along microtubule tracks, and in doing so generate force for the transport of cellular cargo and mitosis. The goal of this project was to define the microtubule interactions and regulation of two kinesin-like proteins (KLPs), the Homo sapiens mitotic kinesin-like protein-1 (HsMKLP-1) and the Arabidopsis thaliana kinesin-like calmodulin-binding protein (KCBP). Functional domains of HsMKLP-1 and KCBP were heterogeneously expressed in insect cells (HsMKLP-1) and/or E. coli (HsMKLP-1, KCBP) and used to examine the microtubule binding and ATPase activity of HsMKLP-1 and KCBP catalytic domains. Overall, the HsMKLP-1 catalytic domain was found to operate in a similar fashion to other KLPs with respect to microtubule binding and ATP hydrolysis, but HsMKLP-1 exhibited enhanced microtubule binding of the dimer and weaker affinity for ATP that functionally distinguishes it from other KLPs. HsMKLP-1 proteins were also used to generate HsMKLP-1 specific antibodies to be used as a tool for characterizing native HsMKLP-1. To define the role of nuclear localization in regulating the activity of HsMKLP-1 during interphase, sequences directing nuclear localization of HsMKLP-1 were identified. Mutation of the nuclear localization sequence 799PNGSRKRR806 to 799PNGSRTSR806 or removal of AA's 830-856 of HsMKLP-1, which contains the nuclear localization sequence 851PKRKKP856, were sufficient to abolish nuclear localization. In the absence of a functional nuclear localization sequence HsMKLP-1 localized to microtubule plus ends, suggesting that nuclear localization serves to limit the interaction of HsMKLP-1 with the interphase microtubule array. The KCBP catalytic domain, which contains a calmodulin-binding site, was used to determine the effect of Ca2+/calmodulin on the microtubule binding and ATPase activity of KCBP. Ca2+/calmodulin was found to inhibit the binding of KCBP to microtubules and reduced the motor's microtubule-stimulated ATPase activity, which suggests that Ca2+/calmodulin may modulate the activity of KCBP in vivo by regulating the motor's association with microtubules.
Ph. D.

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30

Timón, Pérez Krystal 1987. "Spindle assembly and the control of microtubule nucleation through NEDD1 phosphorylation." Doctoral thesis, Universitat Pompeu Fabra, 2018. http://hdl.handle.net/10803/665720.

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During mitosis, the microtubules organize a bipolar spindle that segregates the chromosomes. In higher eukaryotes, these microtubules are nucleated through three different pathways involving the centrosomes (centrosomal pathway), the chromatin (Ran-GTP pathway), and pre-existing microtubules (Augmin-dependent pathway). These three pathways rely on the γ-tubulin ring complex (γ-TuRC) and its adaptor NEDD1. During mitosis, NEDD1 phosphorylation determines its role in microtubule nucleation through the three pathways: Nek9-dependent Ser377 for centrosomal nucleation, AuroraA-dependent Ser405 for the Ran-GTP dependent pathway and Cdk1-dependent Ser411 for the Augmin-dependent microtubule amplification. To define the specific contribution of these microtubule nucleation pathways in spindle assembly, we established several inducible stable cell lines to express phosphorylation variants of NEDD1 on Ser377, Ser405 and Ser411 individually or in combination upon NEDD1 silencing. Our data show that, in agreement with previous results, the three sites are important for spindle assembly. Moreover, they suggest that phosphorylation at Ser411 is responsible for NEDD1 mobility shift in mitosis and plays a major role in NEDD1 function in mitosis.
Durante la mitosis, los microtúbulos se organizan en un huso mitótico que segrega los cromosomas. En organismos eucariotas, estos microtúbulos se nuclean a través de tres vías diferentes que involucran los centrosomas (vía centrosomal), la cromatina (vía dependiente de Ran-GTP/vía cromosomal) y microtúbulos pre-existentes (vía dependiente del complejo proteico Augmin). Estas tres vías dependen de un complejo de nucleación llamado γ-TuRC y la proteína adaptadora NEDD1. Durante la mitosis, la fosforilación de NEDD1 determina su papel en la nucleación de microtúbulos a través de las tres vías: la fosforilación en la Ser377 dependiente de la kinasa Nek9 controla la nucleación centrosomal, la fosforilación en la Ser405 dependiente de la kinasa AuroraA regula la nucleación chromosomal y la fosforilación en la Ser411 dependiente de la kinasa Cdk1 gobierna la nucleación sobre los microtúbulos. Para definir la contribución específica de estas vías de nucleación en el ensamblaje del huso, generamos varias líneas celulares inducibles para expresar mutantes de fosforilación de NEDD1 en Ser377, Ser405 y Ser411 individualmente o en combinación tras silenciar NEDD1 endógena. Nuestros datos muestran que, de acuerdo con resultados previos, los tres sitios de fosforilación son importantes para el ensamblaje del huso. Además sugieren que la fosforilación en Ser411 es responsable del cambio de mobilidad de NEDD1 en mitosis y juega un papel importante en la función de NEDD1 en mitosis.

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Meaders, Johnathan Lee. "Growth, Morphology, and Positioning of Microtubule Asters in Large Zygotes:." Thesis, Boston College, 2020. http://hdl.handle.net/2345/bc-ir:109018.

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Thesis advisor: David R. Burgess
Microtubule (MT) asters are radial arrays of MTs nucleated from a microtubule organizingcenter (MTOC) such as the centrosome. Within many cell types, which display highly diverse size and shape, MT asters orchestrate spatial positioning of organelles to ensure proper cellular function throughout the cell cycle and development. Therefore, asters have adopted a wide variety of sizes and morphologies, which are directly affects how they migrate and position within the cell. In large cells, for example during embryonic development, asters growth to sizes on the scales of hundreds of microns to millimeters. Due to this relatively enormous size scale, it is widely accepted that MT asters migrate primarily through pulling mechanisms driven by dynein located in the cytoplasm and/or the cell cortex. Moreover, prior to this dissertation, significant contributions from pushing forces as a result of aster growth and expansion against the cell cortex have not been detected in large cells. Here we have reinvestigated sperm aster growth, morphology, and positioning of MT asters using the large interphase sperm aster of the sea urchin zygote, which is historically a powerful system due to long range migration of the sperm aster to the geometric cell center following fertilization. First, through live-cell quantification of sperm aster growth and geometry, chemical manipulation of aster geometry, inhibition of dynein, and targeted chemical ablation, we show that the sperm aster migrates to the zygote center predominantly through a pushing-based mechanism that appears to largely independent of proposed pulling models. Second, we investigate the fundamental principles for how sperm aster size is determined during growth and centration. By physically manipulating egg size, we obtain samples of eggs displaying a wide range of diameters, all of which are at identical developmental stages. Using live-cell and fluorescence microscopy, we find strong preliminary evidence that aster diameter and migration rates show a direct, linear scaling to cell diameter. Finally, we hypothesize that a collective growth model for aster growth, or centrosome independent MT nucleation, may explain how the sperm aster of large sea urchin zygotes overcomes the proposed physical limitations of a pushing mechanism during large aster positioning. By applying two methods of super resolution microscopy, we find support for this collective growth model in the form of MT branching. Together, we present a model in which growth of astral MTs, potentially through a collective growth model, pushes the sperm aster to the zygote center
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

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Monda, Julie Kathryn. "Functional analyses of mitotic microtubule-binding complexes." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/119916.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Mitosis is the process by which a single cell divides to form two identical daughter cells. Each daughter cell must inherit a full complement of the genetic material. Thus, a critical aspect of mitosis is the faithful segregation of each duplicated chromosome. Chromosome segregation is achieved through the attachment of a chromosome-localized macromolecular complex, termed the kinetochore, to microtubules. Microtubules are dynamic polymers comprised of tubulin heterodimers. The successful execution of mitosis additionally depends on the organization of the microtubules into a bipolar array, termed the mitotic spindle. The depolymerization of kinetochore-bound microtubules generates the force required to properly segregate the chromosomes. The work in this thesis analyzes the molecular basis for the function and activity of two key players in microtubule function. First, I investigate the mechanisms by which the Ska1 complex facilitates the continued association of the kinetochore with microtubules, even as the microtubules grow and shrink. I show that Ska1 uses multiple surfaces to interact with diverse tubulin substrates, and each of these surfaces are required for microtubule tip tracking and optimal mitotic progression. Second, I analyze cytoplasmic dynein, a microtubule-based motor that is critically required to maintain spindle bipolarity and execute numerous other cellular processes throughout the cell cycle. The execution of these diverse functions of dynein relies on precise temporal and spatial regulation of dynein activity. Dynein regulation is accomplished in part by the association of adaptor proteins with the dynein complex, including Nde1. Here, I show that Nde1 utilizes distinct intermolecular interactions to regulate different dynein functions. I also identify a previously uncharacterized interaction between Nde1 and the 26S proteasome. Finally, I explore a potential role for post-translational modifications in regulating dynein function. I find that the localization of dynein during mitosis is rapidly altered following the addition of small molecule inhibitors of ubiquitination enzymes. Together, these findings provide new insights into the function and regulation of diverse components of the mitotic machinery.
by Julie Kathryn Monda.
Ph. D.

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33

Bendre, Shweta [Verfasser], and Andrea [Akademischer Betreuer] Musacchio. "GTSE1 regulates microtubule stability during mitosis through inhibition of the microtubule depolymerase MCAK / Shweta Bendre ; Betreuer: Andrea Musacchio." Duisburg, 2017. http://d-nb.info/1141053675/34.

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Farache, Dorian. "Etude des fonctions de GCP4, 5 et 6 dans l'assemblage du complexe de nucléation des microtubules." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30231/document.

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Les microtubules sont des composants hautement dynamiques du cytosquelette. La tubuline gamma est localisée au centrosome. Elle y forme le complexe de nucléation des microtubules, le gamma-TuRC, en association avec les protéines GCPs 2-6. Les GCPs 2-6 forment une famille de protéines caractérisée par deux domaines conservés appelés GRIP1 et 2. De par sa structure, le gamma-TuRC sert de moule pour la nucléation des microtubules. Le gamma-TuRC est constitué de plusieurs sous-complexes : les gamma-TuSCs qui sont composés d'une GCP2 et d'une GCP3 qui interagissent entre elle par leur domaine amino-terminal, chacune liant une tubuline gamma via leur domaine carboxy-terminal. Les gamma-TuSCs s'assemblent latéralement pour former une structure à un tour d'hélice, les deux extrémités de l'hélice se recouvrant. La structure atomique de GCP4 s'intègre particulièrement bien dans structure du gamma-TuSC de levure, obtenue en microscopie électronique, à la place de GCP2 et 3 suggérant une forte conservation structurale entre les GCPs. GCP4, 5 et 6 pourraient donc être partie intégrante de l'hélice. Durant ma thèse j'ai étudié la position relative des GCP4, 5 et 6 au sein du gamma-TuRC. Pour cela j'ai développé des approches d'échange de domaines et de mutagénèse. J'ai également mis en place des stratégies de FLIM-FRET et d'immunoprécipitation. J'ai ainsi montré que c'est le domaine N-terminal des GCPs qui définit leur identité, les domaines C-terminaux étant échangeables. J'ai également mis en évidence, au sein du gamma-TuRC, des interactions latérales entre GCP4 et GCP5 semblables à celles établies par GCP2 et GCP3 dans les gamma-TuSC. J'ai également pu isoler un complexe contenant GCP4, 5, 6 et la tubuline gamma indépendamment du gamma-TuRC. J'apporte ainsi les premières preuves expérimentales soutenant l'idée que GCP4, 5 et 6 sont partie intégrante de l'hélice du gamma-TuRC et qu'elles y forment un sous complexe qui occupe une position bien définie
Microtubules are highly dynamic components of the cytoskeleton. gammatubulin is found at the centrosome where it forms a microtubule nucleation complex together with GCPs 2-6, the gamma-TuRC. GCPs 2-6 form a conserved family of proteins characterised by two conserved domains called GRIP1 and 2. The gamma-TuRC functions as a structural template for microtubule nucleation. The gamma-TuRC is composed of smaller subcomplexes called gamma-TuSC. Each gamma-TuSC is composed by one GCP2, one GCP3 and two gamma?tubulins. GCP2 and GCP3 interact via their N-terminal domain and bind gamma tubulin through their C-terminal domain. Several gamma-TuSCs can assemble laterally to form a one-turn helix with the two ends overlapping. The atomic structure of GCP4 fits almost perfectly in the place of GCP2 and GCP3 within the gamma-TuSC envelope obtained by electron microscopy suggesting a strong structural conservation among GCPs. Hence, GCP4, 5 and 6 may be part of the helix. During the course of my thesis, I studied the relative position of GCPs 4, 5, 6 within the gamma-TuRC. To this aim, I developed a domain swapping and mutagenesis approaches. I also combined FLIM-FRET and immunoprecipitation strategies. I have been able to show that the N-terminal domains of GCPs define their identity while the C-terminal domains can be swapped. My results also indicate that GCP4 and GCP5 establish gamma-TuSC like interactions within the gamma-TuRC. I also isolated a complex containing GCP4, 5, 6 and gamma tubulin independently of the gamma-TuRC. My thesis provides the first experimental evidence supporting the model where GCP4, 5 and 6 are part of the gamma-TuRC helix where they form a sub-complex localised at a defined position

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Maney, Robert Todd. "A functional analysis of mitotic centromere-associated kinesin /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/10532.

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Montgomery, Jessica M. "Nek6 controls mitotic progression through regulating EML3 localisation to spindle microtubules." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/37774.

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EMLs are a highly conserved family of microtubule-associated proteins that play a role in microtubule stability. In humans there are six EMLs. EML1, EML2, EML3 and EML4 consist of a largely unstructured basic N-terminal domain (NTD) that contains a short coiled-coil mediating trimerisation, and a highly structured C-terminal domain (CTD) named the TAPE domain. Microtubule binding is conferred through the NTD whilst the TAPE domain binds to soluble tubulin dimers. EML5 and EML6 lack the N-terminal region but have three continuous TAPE domains encoded within a single polypeptide. Previous proteomic studies had identified EML3 as a binding partner of Nek6, a serine/threonine kinase that promotes mitotic spindle assembly. In this study, we have explored the microtubule binding properties of EML3 and its regulation by Nek6. Using a stable cell line expressing YFP-EML3, fixed and time lapse imaging revealed that EML3 associates along the length of microtubules and exhibits rapid recovery following photobleaching. Whilst microtubule binding of the related EML1-NTD was reduced upon incubation of microtubules with the protease subtilisin in vitro. This suggests an electrostatic interaction between the basic EML-NTD and the acidic tubulin C-terminal tails. Immunoprecipitation experiments confirmed the interaction between EML3 and Nek6 and revealed that this interaction is increased in mitosis. Unexpectedly, while EML3 bound strongly to interphase microtubules, it exhibited much lower affinity for microtubules in mitosis. Depletion of Nek6 led to accumulation of EML3 on spindle microtubules in mitosis, whilst expression of a constitutively active Nek6 displaced EML3 from interphase microtubules. By mass spectrometry a number of phosphorylation sites were identified within the basic NTD of EML3 upon incubation with Nek6. Furthermore, introduction of acidic charge into the basic NTD of EML3 perturbed its association with interphase microtubules. Hence, we propose that phosphorylation of EML3 by Nek6 disturbs electrostatic interactions between the basic N-terminus and the acidic surface of MTs. This leads to reduced association of EML3 with microtubules in mitosis. We speculate that this decreases microtubule stability, facilitating mitotic spindle assembly and search and capture of condensed chromosomes.

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Espeut, Julien. "Régulation de la motilité de Cenp-E, une kinésine associée au kinétochore." Montpellier 2, 2007. http://www.theses.fr/2007MON20133.

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Le point de contrôle de l'assemblage du fuseau mitotique va permettre d'empêcher la division cellulaire en cas de mauvais positionnement des chromosomes sur la plaque métaphasique. Parmi les nombreuses protéines impliquées dans cette régulation se trouve la protéine du kinétochore Cenp-E (Centromere associated protein E). Un des enjeux majeur dans l'étude de la mitose est de déterminer quelles sont les protéines du kinétochore qui sont responsables du mouvement des chromosomes. L'objectif de ma thèse a alors été de déterminer quels étaient les mécanismes de régulation de l'activité motrice de la kinésine Cenp-E impliqués dans la dynamique de liaison des microtubules aux kinétochores. Nous avons pu déterminer que la protéine Cenp-E se déplace vers l'extrémité "+" des microtubules. De plus, la partie C-terminale de Cenp-E inhibe directement son activité motrice et cette inhibition est reversée par une phosphorylation par les kinases Mps1 et Cdk1-cycline B. Ces résultats suggèrent un contrôle dynamique de la motilité de Cenp-E et de la congression des chromosomes, dépendant d'une phosphorylation au kinétochore
The mitotic spindle assembly checkpoint stops cell division in case of incorrect positioning of the chromosomes onto the metaphase plate. This checkpoint avoids inaccurate chromosome division, which could result in a loss or gain of genetic material. Amongst the many proteins involved in this regulation is the kinetochore protein Cenp-E (Centromere associated protein E). One of the biggest goal in the field of mitosis is to establish which kinetochore proteins are responsible of chromosome movement. Therefore the aim of my thesis has been to determine the mechanisms of regulation of the Cenp-E kinesin motor activity involved in the dynamics of binding to the microtubules. We have been able to show that Cenp-E moves towards the "+" end of microtubules. Furthermore, the C-terminal portion of Cenp-E directly inhibits its motor activity and such inhibition is reversed by phosphorylation of the Cterminus by the kinases Mps1 and Cdk1-cyclin B. These results suggest a dynamic control of Cenp-E motility and of chromosome congression, dependent on kinetochore phosphorylation

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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 defects
La 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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Holmfeldt, Per. "Regulation of tubulin heterodimer partitioning during interphase and mitosis." Doctoral thesis, Umeå : Department of Molecular Biology, Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1923.

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40

Caudron, Maïwen. "Coordination of mitotic spindle assembly by chromosome-generated molecular interaction gradients." Université Louis Pasteur (Strasbourg) (1971-2008), 2005. http://www.theses.fr/2005STR13056.

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Au cours du cycle cellulaire, les chromosomes sont distribués une seule fois et de façon identique entre les deux cellules filles. Ceci est effectué par le fuseau mitotique, une machine complexe composée de nano tubes de protéines orientés, les microtubules. Ces tubes s'auto organisent en une structure bipolaire au sein de laquelle les chromosomes se positionnent sur l'équateur et interagissent avec les microtubules au niveau de structures spécialisées, les kinétochores. Au début de la division cellulaire, les microtubules qui étaient alors longs et stables deviennent brusquement plus courts et dynamiques. Cela est dû à un changement général d'état du cytoplasme. Il a été observe qu'en l'absence de chromosomes, les microtubules ne pouvaient pas s'auto organiser en un fuseau mitotique bipolaire au sein du cytoplasme mitotique. Cette observation surprenante a soulevé la question des mécanismes impliqués dans le processus d'auto organisation du fuseau mitotique et en particulier du rôle possible des chromosomes. Ceci était intéressant dans la mesure où cela représenterait un exemple de coordination de l'assemblage d'une machine par l'objet même sur lequel elle agit. Il s'est avéré que les chromosomes jouent effectivement un rôle central dans le processus de formation du fuseau mitotique, en modifiant localement l'état du cytoplasme, ce qui induit la nucléation et la stabilisation de microtubules. Les résultats présentés ici montrent théoriquement et expérimentalement que les chromosomes génèrent des gradients d'interactions moléculaires qui procurent une information spatiale nécessaire à la coordination de la nucléation et de la stabilisation des microtubules, deux événements essentiels dans le processus d'auto organisation du fuseau mitotique. Une petite molécule que l'on nomme Ran, existe sous deux formes. Un état riche en énergie qui contient du GTP et un état pauvre en énergie qui contient du GDP. Il y a un facteur sur les chromosomes qui échange le GDP de Ran pour du GTP et un autre facteur dans le cytoplasme qui active l'activité GTPasique de Ran. Ceci conduit en principe à une haute concentration locale en Ran GTP autour des chromosomes. Sous cette forme, Ran interagit avec des complexes moléculaires présents dans le cytoplasme. Ces complexes sont appelés importine-protéines nucléaires (protéines à NLS). Lors de la liaison du Ran GTP aux importunes, les protéines NLS sont relâchées autour des chromosomes. Il se trouve que parmi ces protéines, il y a des molécules qui induisent la nucléation et la stabilisation des microtubules. Dans ma thèse, j'ai montré que de tels effets sont importants pour l'assemblage du fuseau mitotique. Ensuite, j'ai montré qu'il était possible de calculer la forme des gradients de RanGTP libre, de complexes Ran GTP importines et de proteins-NLS libres en utilisant les équations de reaction-diffusion. Finalement, j'ai visualisé le gradient formé par le complexe Ran GTP-importin en utilisant des méthodes de FLIM et montré que ce gradient pouvait être lu de façon spécifique par le système microtubulaire de telle sorte qu'il induise la nucléation des microtubules près des chromosomes et leur stabilisation à plus grande distance. En résumé, j'ai montré que des phénomènes de reaction-diffusion autour des chromosomes sont à la base du contrôle de l'auto organisation des microtubules en un fuseau bipolaire
Once in every cell cycle, living cells distribute evenly their chromosomes to the two daughter cells. The cellular machine that achieves chromosome segregation is the mitotic spindle, which is made of oriented protein nanotubes arranged into a bipolar system that surround the chromosomes to which the tubes are attached through specialized regions, the kinetochores. At the onset of cell division, microtubules that were long and stable suddenly become shorter and highly dynamic. This is due to a general change in the state of the cytoplasmic environment. Surprisingly, the mitotic cytoplasm does support the assembly of the bipolar spindle in the absence of chromosomes, raising questions about the mechanism of spindle assembly and the role of chromosomes in this process. This was interesting since this could represent an example of the coordination of the assembly of a machine by the very substrate on which it is acting. It appeared that chromosomes actually play a central role in spindle assembly by modifying locally the nature of the cytoplasm in their vicinity, thereby promoting the nucleation and stabilization of microtubules that finally self-organize into a bipolar array thanks to the action of various molecular motors. In this thesis, I show both theoretically and experimentally that chromosomes generate gradients of molecular interactions that provide spatial cues required for the coordination of microtubule nucleation and plus end stabilization two essential events in the pathway that leads to the self-organization of the mitotic spindle. A small molecule called Ran can be present in two forms. A high-energy state that contains GTP and a low energy state that is loaded with GDP. On the chromosomes, there is an exchange factor that loads Ran with GTP and in the cytoplasm there is a GTPase “activating factor” that forces Ran to change the bound GTP into GDP. Previous work had shown that the local production of Ran GTP around chromosomes leads to its interaction with molecular complexes present in cells. These complexes are called Importin- [Nuclear-Localization-Signal-containing proteins] (NLS-proteins). Upon binding of Ran GTP to importins, NLS-proteins are released around chromosomes. It turns out that among these NLS-proteins, there are molecules that trigger microtubule nucleation and stabilize microtubule plus ends when they are released from importins. In my thesis, I have shown that such a local effect on microtubule nucleation and stabilization is important for the proper formation of a mitotic spindle. Then, I showed that reaction-diffusion equations allow calculating the shape and extent of the gradients of various molecular species like free Ran GTP, Ran GTP-importin complexes and free NLS-proteins. Finally, I have used Fluorescence Life time Imaging (FLIM) technology to visualize the shape of the Ran GTP-importin gradient and demonstrated that this gradient was read differently by the microtubule system so that microtubule nucleation would occur close to chromosomes while stabilization effects would be sensed much further away. In summary, I have shown that a reaction-diffusion process occurring around chromosomes governs important aspects of the self-organization of microtubules into a bipolar spindle

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41

Perchey, Renaud. "Le rôle de p27Kip1 dans la mitose et dans l'instabilité génétique." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30055.

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L'avancée du cycle cellulaire est régulée par l'activation séquentielle des complexes cycline/CDK. Un niveau de régulation de ces complexes est médié par leur association avec leurs inhibiteurs, les CKIs dont p27Kip1 (p27). p27 est donc un inhibiteur du cycle de division cellulaire, ce qui lui confère un rôle de suppresseur de tumeur. Cependant, dans certains contextes, notamment lorsque p27 est localisé dans le cytoplasme, p27 a des fonctions oncogéniques. En effet, en plus de ses rôles dans la régulation des complexes cycline/CDK, p27 possède de multiples rôles CDK-indépendants et est impliqué dans le contrôle de divers processus comme la migration, la transcription, l'autophagie et la cytocinèse. La détermination de l'intéractome de p27 a montré que plusieurs protéines jouant des rôles clés dans la cytocinèse peuvent interagir avec p27, dont PRC1 (Protein Regulating Cytokinesis 1) et Citron-Kinase. Les objectifs de ma thèse sont de comprendre comment p27 participe au contrôle de la cytocinèse via son interaction avec ces deux protéines. Régulation de l'activité de PRC1 par p27Kip1 PRC1 est nécessaire à la formation du fuseau central en début d'anaphase via son interaction avec les microtubules et d'autres partenaires. Pour ce projet, mes objectifs étaient de confirmer l'interaction de PRC1 avec p27, de cartographier les domaines d'interactions, de déterminer si p27 pouvait réguler l'activité de PRC1 et par quel mécanisme, et enfin les conséquences de cette régulation au niveau cellulaire. J'ai confirmé l'interactions de p27 avec PRC1 dans différents modèles cellulaires et identifié les domaines d'interactions respectifs. J'ai mis en évidence par une technique de sédimentation des microtubules assemblés in vitro que p27 empêche l'interaction de PRC1 avec les microtubules. J'ai pu mettre en évidence deux phénotypes. D'une part la surexpression de PRC1 induit une fasciculation excessive et massive des microtubules qui est abolie par la co-expression de p27. Par ailleurs, la surexpression de PRC1 induit la binucléation et la co-expression de p27 empêche la binucléation induite par PRC1. Ce phénotype est révélateur d'un échec de la cytocinèse et suggère un rôle CDK-indépendant de p27 durant cette phase. Régulation de Citron-Kinase par CDK1 et p27Kip1 Nous avions déjà démontré que p27 interagit avec Citron-Kinase (CitK). CitK a un rôle essentiel dans la cytocinèse, sa déplétion induit un échec de cytocinèse. CitK est impliqué dans les dernières étapes de la cytocinèse (abscission), notamment en tant que protéine de structuration permettant de faire un lien entre la membrane plasmique et l'anneau contractile. Les mécanismes de régulation de CitK restent mal connus et il est important de comprendre comment cette protéine essentielle pour le déroulement de la cytocinèse est régulée. Dans notre étude de 2012, un mutant p27CK-, qui n'interagit pas avec les complexes cycline/CDK et n'est pas dégradé correctement par le protéasome, induit un phénotype de multinucléation en interférant avec l'activité de CitK, notamment via la régulation de l'interaction CitK/RhoA. Nous pensions initialement que l'accumulation de p27CK- en G2/M était responsable de ce phénotype
Progression through the cell cycle is regulated by the sequential activation of cyclin/CDK complexes. One level of regulation of these complexes is provided by their association with their inhibitors, the CKIs, which include p27Kip1 (p27). p27 is therefore an inhibitor of the cell division cycle, in this way, p27 acts as a tumor suppressor. However, in certain contexts, and especially when p27 is located in the cytoplasm, p27 has oncogenic functions. It appears that in addition to its role in the regulation of cyclin/CDK complexes, p27 has multiple CDK-independent roles and is involved in the control of various cellular processes such as migration, transcription, autophagy and cytokinesis. Determination of the interactome of p27 revealed that several proteins playing an essential role in cytokinesis could interact with p27, including PRC1 (Protein Regulating Cytokinesis 1) and Citron-Kinase (CitK). The objectives of my thesis are to understand how p27 participates in the control of cytokinesis via its interaction with these two proteins. Regulation of PRC1 activity by p27Kip1 PRC1 is necessary for the formation of the central spindle at the beginning of anaphase via its interaction with microtubules and other partners. For this project, my objectives were to confirm the p27/PRC1 interaction, to map the interaction domains on each partner, to test whether p27 could regulate PRC1 activity and determine by which mechanism, as well as the cellular consequences of this regulation. I validated the interactions in different cells models and identified the respective interaction domains. I found by a microtubule sedimentation technique in vitro that p27 prevents the interaction of PRC1 with microtubules. I was able to uncover two phenotypes. First, PRC1 overexpression induces excessive and massive microtubule bundling, which is abolished by co-expression of p27. Second, PRC1 overexpression causes binucleation and p27 co-expression prevents PRC1-induced binucleation. This phenotype is indicative of cytokinesis failure and suggests a CDK-independent role of p27 during cytokinesis. Citron-Kinase regulation by CDK1 and p27Kip1 Another p27 interactor previously reported by my team is CitK. CitK plays an essential role in cytokinesis and its depletion induces cytokinesis failure. CitK is involved in the final stages of cytokinesis (abscission), notably as a scaffold protein that bridges the plasma membrane and the contractile ring. The regulatory mechanisms of CitK remain poorly understood and it appears important to understand how this protein playing an essential role in cytokinesis is regulated. In our 2012 study, a p27CK- mutant, that cannot bind and inhibit cyclin-CDK complexes and is not degraded correctly by the proteasome, induced a phenotype of multinucleation by interfering with CitK activity, notably via the regulation of the CitK/RhoA interaction. We initially thought that the accumulation of p27CK- in G2/M drove this phenotype. However, our current data suggest that although the total amount of p27CK- is elevated, the amount of p27CK- at the midbody are similar to that of p27WT, suggesting that p27CK- lacks a feature that p27WT has. Since the only difference between p27WT and p27CK- is the ability to bind and inhibit cyclin/CDK complexes, we hypothesized that CitK can be regulated by CDK1 and that p27 participates in this regulation. My work has shown that CitK interacts with CDK1/Cyclin B1 in different cells lines

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42

Mathieu, Michelle. "Regulation of mitotic BubR1 phosphorylation by the BubR1 pseudokinase domain." Master's thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/27062.

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BubR1 est une protéine importante dans le point de contrôle de la mitose pour la stabilisation des interactions entre kinétochores et microtubules (KT-MT). Ces fonctions protègent de la ségrégation anormale des chromosomes et de l’instabilité du génome. BubR1 possède des sites de phosphorylation mitotique hautement conservés dans le domaine régulant l’attachement des kinétochores (KARD), où S676 et S670 sont phosphorylées respectivement par la kinase polo-like 1 (Plk1) et par la kinase cycline-dépendante 1 (Cdk1). Ces sites de phosphorylation sont essentiels pour le recrutement de la phosphatase PP2A-B56, qui stabilise les interactions KT-MT. Nos résultats montrent que la délétion entière ou des mutations qui déstabilisent le domaine pseudokinase de BubR1, causent la perte de phosphorylation des résidus S676 et S670 en mitose. Notre hypothèse est que le domaine pseudokinase de BubR1 peut jouer un rôle essentiel dans la régulation de la phosphorylation du KARD et donc dans la stabilisation des interactions KT-MT.
The mitotic protein BubR1 functions in the spindle assembly checkpoint (SAC) by stabilizing kinetochore-microtubule (KT-MT) interactions. These functions protect the cell from abnormal chromosome segregation and genome instability. BubR1 has highly conserved mitotic phosphorylation sites in the kinetochore-attachment regulatory domain (KARD); the residue S676 is phosphorylated by polo-like kinase-1 (Plk1) and S670 is phosphorylated by cyclin-dependent kinase-1 (Cdk1). These phosphorylation sites are essential for KARD recruitment of protein phosphatase PP2A-B56, which stabilizes KT-MT interactions. Our results show that mutations that cause pseudokinase domain instability and a highly stable truncation mutant of BubR1 were found to cause loss of mitotic S676 and S670 phosphorylation. We hypothesize that the pseudokinase domain of BubR1 may play an important role in the regulation of KARD phosphorylation and thus the stabilization of KT-MT interactions.

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Gouveia, Susana Montenegro. "Funcional analysis of microtubule binding domain of MAST." Master's thesis, Universidade de Aveiro, 2005. http://hdl.handle.net/10773/4979.

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Mestrado em Métodos Biomoleculares Avançados
A Mast/Orbit/CLASP é uma família conservada de proteínas associadas aos microtúbulos (MAPs) essenciais para a organização e função do fuso mitótico (Inoue et al., 2000; Lemos et al., 2000; Akhmanova et al., 2001; Maiato et al., 2002; Maiato et al., 2003a; Mimori-Kiyosue et al., 2005). Estas proteínas surgem associadas aos microtúbulos, centrossomas e cinetocoros e diversos estudos sugerem que desempenham um papel importante na regulação das propriedades dinâmicas dos microtúbulos (Akhmanova et al., 2001; Maiato et al., 2002; Maiato et al., 2003a; Maiato et al., 2005). As isoformas humanas, CLASPs, fazem parte de um conjunto de proteínas (+TIPs) que exibem uma forte acumulação na ponta de crescimento (+) dos microtúbulos em polimerização (Schuyler and Pellman, 2001). Estas proteínas dissociam-se do polímero formado o que origina uma localização em forma de cometa na extremidade do microtúbulo. Neste trabalho mostramos que em Drosophila, a proteína Mast também é uma +TIP. Adicionalmente, definimos o domínio de ligação da proteína aos microtubulos e demonstrámos que, in vitro, a Mast se associa directamente com a tubulina num processo sensível a nucleótidos de guanina. O GTP favorece a ligação aos heterodímeros de tubulina, mas não influencia a ligação aos microtubulos. Contrariamente, o GDP inibe fortemente a ligação da Mast aos microtúbulos e heterodímeros de tubulina. Finalmente, provamos que a Mast liga e hidrolisa GTP, o que a torna a primeira +TIP com características de GTPase e sugere um novo mecanismo para a localização dinâmica das +TIPs. Estes resultados são consistentes com um modelo no qual a Mast-GTP copolimeriza com os heterodímeros de tubulina ou se associa directamente à extremidade (+) dos microtúbulos em crescimento. Após a associação ao microtúbulo dá-se a hidrólise do GTP e consequente formação de Mast-GDP que causará uma alteração conformacional da proteína promovendo a sua dissociação do microtúbulo. Este estudo sugere que uma proteína associada aos microtúbulos pode utilizar a actividade GTPásica na regulação da sua ligação aos microtúbulos.
Mast/Orbit/CLASP is a conserved MAP protein family essential for the organization and function of mitotic spindle (Inoue et al., 2000; Lemos et al., 2000; Akhmanova et al., 2001; Maiato et al., 2002; Maiato et al., 2003a; Mimori-Kiyosue et al., 2005). It accumulates at centrosomes, kinetochores and microtubule plus-ends where it is thought to regulate their dynamic properties (Akhmanova et al., 2001; Maiato et al., 2002; Maiato et al., 2003a; Maiato et al., 2005; Mimori-Kiyosue et al., 2005). CLASPs, the human homologues (Akhmanova et al., 2001), are members of the microtubule plus-end tracking protein (+TIP) family (Schuyler and Pellman, 2001). +TIPs show strong accumulation at the polymerizing end of microtubules, dissociating from the polymer soon afterwards giving the appearance of a comet-like structure. Here we show that the Drosophila homologue Mast also displays +TIP behaviour. Moreover, we defined the microtubule binding domain of Mast and showed that it associates directly with tubulin in a guanine nucleotide sensitive manner. GTP favours the binding of Mast to tubulin heterodimers but does not influence binding to microtubules, while GDP strongly inhibits the binding of Mast to microtubules. More importantly, we show that Mast can bind and hydrolyse GTP demonstrating that it is the first +TIP with this feature and hence suggesting a new mechanism for +TIP behaviour. These results are fully consistent with a model in which Mast-GTP copolymerizes with tubulin heterodimers at the growing microtubule plus end. Mast is then released from the polymer due to hydrolysis of the bound GTP, causing a conformational change of the protein that promotes its release from the microtubule lattice. Our data provides evidence that a microtubule associated protein could use its GTPase activity to regulate its ability to bind microtubules.
FCT
POCTI/BCI/49176/2002

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44

Conti, Duccio. "Role of phosphatases in the end-on conversion process." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/278657.

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Proper attachment of chromosomes to microtubules is important for the accurate segregation of chromosomes and genome stability. The initial engagement of chromosomes happens along the lateral wall of microtubules through a highly specialised protein structure assembled on the centromeric DNA, the kinetochore. Ultimately, kinetochores must be attached to the ends of microtubules (a geometry called end- on attachment). A series of highly dynamic steps called the end-on conversion process, converts the initial immature lateral attachments into mature end-on attachments. How this process is finely tuned by phosphorylation and dephosphorylation to achieve stable attachments is still unclear. Furthermore, what is the role of microtubule-associated proteins in the stabilisation of kinetochore-microtubule attachments is unknown. This project aimed to study the role of phosphatases in the regulation of the end-on conversion process. First, I investigated the different contribution of the two outer-kinetochore phosphatases - BubR1- recruited PP2A-B56 and KNL1-recruited PP1 - in counteracting Aurora B kinase during the end-on conversion process. I found that BubR1-recruited PP2A-B56 plays an essential role in the process, but KNL1-recruited PP1 does not. I also investigated whether the HEC1/Ndc80 N-tail is a critical substrate of Aurora B phosphorylation for the stabilisation of the end-on attachments. Using a phospho-dead mutant of the HEC1/Ndc80 N-tail, I discovered that cells are still susceptible to Aurora B activity, indicating downstream pathways independent of HEC1/Ndc80. Then, I studied the biological role of the Astrin C-terminus, where an evolutionarily conserved RVMF motif, a putative PP1 binding site, is located. My findings show C-terminal Astrin mutants fail to localise at kinetochores of both monopolar and bipolar spindles; induce defects in the end-on conversion process in monopolar spindles and prolong mitosis time with increased Mad2 levels at the outer-kinetochore. A kinase inhibitor assay showed that kinetochore-microtubule attachment defects in Astrin mutant expressing cells could be rescued when both Aurora B and Cdk1 kinases are inhibited, suggesting a role for Astrin’s C-terminus in counteracting Aurora B and Cdk1 activity. Finally, I probed the putative interaction of the Astrin C-terminus and PP1 using biochemistry, cell biology and fluorescence microscopy techniques. I discovered that artificially targeting PP1 onto the Astrin C-terminus but not on the N-terminus rescues mutants localisation defects at the kinetochore. In summary, my results indicate that Astrin and PP1 interact at the kinetochore of living cells. In conclusion, my work shows that mitotic phosphatases have distinctive contributions in the regulation of the dynamic steps of the end-on conversion process and that Astrin is a potential PP1 phosphatase recruiter at the outer-kinetochore, where is necessary for the stabilisation of end-on attachments.

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45

Lioutas, Antonio 1980. "Aurora A kinase function during anaphase." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/97290.

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Aurora A (AurA) is an important mitotic kinase mainly studied for its involvement in cell cycle progression, centrosome maturation, mitotic spindle pole organization and bipolar spindle formation. It localizes to duplicated centrosomes and spindle microtubules (MTs) during mitosis where it regulates various factors participating in metaphase spindle formation. AurA is degraded late in mitosis suggesting that it might also have a function in anaphase. In this study we focused in understanding AurA function during anaphase in two different experimental systems. First, we kept AurA active in cycled Xenopus egg extracts and found that MTs maintained their mitotic organization longer throughout mitotic exit. We also observed chromosome segregation defects and problematic nuclear envelope formation. These observations indicate that AurA activity needs to be down-regulated for the transition from metaphase back to interphase. To get insights into the role of AurA during metaphase-anaphase transition we initially asked whether its kinase activity is still necessary for the maintenance of the metaphase spindle. We saw that the inhibition of AurA kinase activity in metaphase resulted to a collapse of the established metaphase spindle in HeLa cells. Indicating that AurA activity is necessary for the metaphase spindle maintenance. Then, we looked whether AurA kinase activity is still necessary during anaphase. We inhibited AurA at the onset of anaphase in Hela cells and found that anaphase spindles were smaller. We also observed that the MT structure responsible for anaphase spindle elongation, the central spindle, was defectively assembled and organized. Moreover, in cells where AurA was inhibited segregation of chromosomes was defective. These results indicate that AurA kinase activity is necessary for anaphase spindle elongation, central spindle assembly and organization and chromosome segregation. To understand further how AurA regulates anaphase spindle formation we looked known AurA substrates. We depleted TACC3, a known AurA substrate involved in MT formation earlier in mitosis and observed that TACC3 depletion phenocopied AurA inhibition. This indicates that TACC3 has a function in MT organization and chromosome segregation during anaphase and this function could possibly be regulated by AurA. In this study we have demonstrated that AurA activity is essential for metaphase spindle maintenance. We also found that during anaphase when AurA is either maintained active or inhibited MT organization is greatly affected and chromosome segregation is defective. Suggesting that AurA activity needs to be tightly controlled during anaphase for a correct completion of mitosis.
Aurora A (AurA) es una quinasa mitótica importante que se ha estudiado principalmente en su papel durante la progresión del ciclo celular, la maduración del centrosoma, la organización y la formación del polo y del huso mitótico. Durante la mitosis, AurA se localiza en los centrosomas duplicados y en los microtúbulos (MTs) del huso y se ha observado que regula varios factores que participan en la formación del huso mitótico. AurA se degrada al final de la mitosis indicando que pueda tener una función durante la anafase. En este estudio nos hemos centrado en la comprensión de la función de AurA durante la anafase en dos sistemas experimentales diferentes. En primer lugar, utilizando extractos de huevos de Xenopus hemos mantenido AurA activa durante la transición de metafase a anafase y hemos visto que los MTs del huso mitótico mantienen su organización durante más tiempo. También hemos observado que cuando AurA se mantiene activa existen defectos en la segregación cromosómica y la formación de la membrana nuclear. Esto indica que la actividad de AurA tiene un papel regulador sobre los MTs y la chromatina durante la transición de la metafase a la interfase. Para entender cual es la función de AurA durante la transición de metafase a anafase primero hemos estudiado si la actividad de la quinasa es necesaria para el mantenimiento del huso mitótico. Hemos visto que la inhibición de la actividad quinasa AurA resultó en el colapso del huso durante la metafase en células HeLa. Esto indica que la actividad de AurA es necesaria para el mantenimiento del huso mitótico de metafase. A continuación hemos analizamos si la actividad quinasa de AurA sigue siendo necesaria para la anafase. Para ello hemos inhibido AurA en células Hela al inicio de la anafase. En estas condiciones los husos de la anafase son más pequeños y la estructura de los MTs responsable del alargamiento del huso mitótico durante la anafase, el huso central, se organiza defectuosamente. Además, se encontraron errores durante la segregación de los cromosomas. Estos resultados indican que la actividad quinasa de AurA es necesaria para el alargamiento del huso durante la anafase y la organización y segregación cromosómica. Para entender el mecanismo de la función de AurA durante la anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 , un sustrato conocido de AurA que participa en la formación de MTs en las fase iniciales de la mitosis hemos encontrado que su eliminación de células HeLa produce el mismo fenotipo que la inhibición de AurA. Esto indica que TACC3 tiene una función en la organización de MT y la segregación de cromosomas durante la anafase y que esta función podría estar regulada por la quinasa AurA. En este estudio hemos demostrado que la actividad quinasa de AurA es esencial para el mantenimiento del huso mitótico. También hemos encontrado que durante la anafase cuando la quinasa AurA se mantiene activa o se inhibe la organización de los MTs del huso mitótico se ve muy afectada y los cromosomas se segregan defectuosamente. Por tanto los resultados de este estudio indican que la actividad quinasa de AurA está estrechamente controlada durante la anafase para el correcto cumplimiento de la mitosis.

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46

Kim, Yumi. "Mechanism of the mitotic kinesin CENP-E in tethering kinetochores to spindle microtubules." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3369626.

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Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed September 15, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 109-127).

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47

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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Schütz, Martin Maximilian. "The role of NIMA-like kinase Nek9 in mitosis." Doctoral thesis, Universitat Pompeu Fabra, 2011. http://hdl.handle.net/10803/38705.

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Mitosis is the essential process during which a cell divides into two viable daughter cells. To allow a faithful segregation of the chromosomes into each daughter, the cell forms the bipolar spindle. The NIMA-like kinase family member Nek9 has been previously proposed to play a role in bipolar spindle assembly and in the chromosomal pathway of microtubule assembly. We aimed at gaining a better understanding of Nek9 function by characterizing Xenopus Nek9, xNek9, using the Xenopus egg extract system. We have shown that xNek9 may not act through the kinase cascade xNek9 – xNek6 in meiosis as described for human Nek9 in somatic cells and therefore may have different substrates. Furthermore, we have demonstrated by depletion, increased addition of Flag-hNek9 and a dominant-negative approach that xNek9 is important for bipolar spindle formation. In addition, we have shown that xNek9 depletion causes decreased microtubule density in bipolar spindles and slower RanGTP induced aster formation. We identified xNedd1, the adaptor protein for the γTuRC, as a novel interactor and substrate of xNek9. xNek9 depletion reduces the recruitment of xNedd1 to sperm nuclei induced aster and decreases the number and length of nucleated microtubules. These data suggest that one role of xNek9 in spindle assembly is exerted through xNedd1 regulation. We propose a model for xNek9 – xNedd1 interaction and a putative mechanism for the regulation of xNek9 – xNedd1 explaining how they fulfil their role in bipolar spindle assembly.
La mitosis es el proceso esencial durante el cual una célula se divide en dos células hijas viables. Para permitir una segregación fiable de los cromosomas en cada hija, la célula forma el huso bipolar. Nek9, el miembro de la familia de quinasas NIMA-like ha sido propuesto para desempeñar un papel en el la asamblea del huso bipolar y en la vía cromosómica de ensamblaje de los microtúbulos. Nuestro objetivo era lograr una mejor comprensión de la función Nek9 caracterizando Nek9 de Xenopus, xNek9, utilizando el sistema de extracto de huevos de Xenopus. Hemos demostrado que xNek9 probablemente no actuará a través de la cascada de las quinasas xNek9 - xNek6 en la meiosis como se describe para Nek9 humano en células somáticas y por lo tanto pueden tener diferentes sustratos. Además, hemos demostrado por el agotamiento, la adición incrementada de Flag-hNek9 y un enfoque dominante-negativas que xNek9 es importante para la formación del huso bipolar. Además, hemos demostrado que el agotamiento xNek9 causa una disminución de la densidad de los microtúbulos en los husos bipolares y una formación más lenta de asteres inducidos por RanGTP. Se identificó xNedd1, la proteína adaptadora para el γTuRC, como un interactor y sustrato novedoso de xNek9. El agotamiento de xNek9 reduce la contratación de xNedd1 a los asteres inducidas por núcleos de espermatozoides y disminuye el número y longitud de microtúbulos nucleados. Estos datos sugieren que un papel de xNek9 en el conjunto del huso se husorce a través de la regulación de xNedd1. Proponemos un modelo para la interacción xNek9 – xNedd1 y un supuesto mecanismo para la regulación de xNek9 – xNedd1 explicando cómo cumplen su papel en la ensamblaje del huso bipolar.

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49

Lacroix, Benjamin. "Rôle de la polyglutamylation dans le contrôle sélectif des moteurs moléculaires et des MAPs." Montpellier 2, 2009. http://www.theses.fr/2009MON20196.

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Abstract:

Les microtubules (MTs) sont des éléments du cytosquelette impliqués dans de nombreux processus cellulaires essentiels comme le transport intracellulaire, la motilité cellulaire ou la division cellulaire. La polyglutamylation est une modification post-traductionnelle présente sur les tubulines alpha- et beta- qui sont les protéines constitutives des MTs. Cette modification est présente sur des résidus glutamate de la partie C-terminale de la tubuline, qui est connue pour être un domaine important dans l'interaction avec les MAPs et les moteurs moléculaire. La polyglutamylation consiste en l'ajout séquentiel d'un nombre variable de résidus glutamates sur la chaîne primaire d'une protéine. Les enzymes responsables de cette modification ont été identifiés au laboratoire comme appartenant à la famille des protéines TTLLs (Tubulin Tyrosine Ligase Like proteins). Au cours de ma thèse j'ai d'abord étudié les propriétés enzymatiques de chaque polyglutamylase. J'ai pu mettre en évidence que chaque enzyme possède une spécificité de substrat (alpha ou beta-tubuline) et une préférence pour l'initiation ou l'élongation de la chaine. Le taux de glutamylation des MTs varie au cours du cycle cellulaire. Les MTs en interphase ne sont pas modifiés alors que le fuseau mitotique et le midbody le sont fortement. Cette localisation spécifique de la modification suggère un rôle particulier au niveau de ces structures. J'ai pu démontrer l'implication de la polyglutamylation dans le recrutement subcellulaire d'une MAP mitotique et dans l'activation de protéines impliquées dans la coupure des MTs, les severing proteins spastine et katanine
Microtubules (MTs) are cytoskeletal filaments that are essential for many different cellular functions including cell division, intracellular transport and cell motility. Polyglutamylation is a post-translational modification that targets alpha- and beta-tubulin, the building blocks of MTs. This modification is present on the C-terminal tail of the tubulins, which is a main domain of interaction between MTs and MAPs (MTs associated proteins). Tubulin polyglutamylation is a complex modification that can generate long side chains of glutamates on a single modified residue of the protein. My first focus was to characterize the enzymatic properties of the polyglutamylases of the family of TTLLs (Tubulin Tyrosine Ligase Like proteins). I found that each enzyme has a specificity toward initiation or elongation of the side chains and for preferentially modifying alpha or beta tubulin. The polyglutamylation level on MTs varies during the cell cycle: interphase MTs are almost not glutamylated whereas spindle and midbody MTs are strongly modified. This suggests that tubulin polyglutamylation plays a specific role during mitosis. I demonstrated that the glutamylation pattern present on mitotic MTs regulates the localisation and activity of MAPs. I found that the mitotic MAP (TPX2) is recruited specifically to glutamylated MTs and that polyglutamylated tubulin activates the MT-severing activity of two proteins, katanin and spastin

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50

Conte, Nathalie [Aude]. "TACC-TIC cellulaires." Aix-Marseille 2, 2003. http://www.theses.fr/2003AIX22079.

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Dissertations / Theses on the topic 'Microtubules. Mitosis'

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