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Wang, Bin. "Phosphoproteome studies of human mitotic spindle proteins." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-123191.
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Plumb, Kemp. "Measuring mitotic spindle dynamics in budding yeast." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86726.
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In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells.In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface.
In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.
La régulation du cycle cellulaire et la prolifération de générations viables requièrent à la fois la reproductibilité et la coordination des différents processus complexes en jeu dans un environnement toutefois dominé par l'agitation thermique. Un exemple essentiel est l'assemblage et la migration du fuseau mitotique qui doivent avoir lieu correctement avant même la ségrégation des chromosomes. Le fuseau mitotique est une structure transitoire composée de deux pôles séparés par des filaments de protéines appelés les microtubules, auxquelles est rattaché le matériel génétique de la cellule. Le fuseau mitotique est notamment impliqué dans l'alignement des chromosomes, puis leur ségrégation vers des pôles opposés de la cellule ; d'où la nécessité d'un positionnement spatial précis et temporellement coordonné du fuseau mitotique pour assurer la division correcte du matériel génétique entre les cellules mère et fille.
Dans ce mémoire, je décrirai les techniques de microscopie confocale par fluorescence ainsi que les algorithmes automatisés d'analyse d'image, que j'ai mis en oeuvre pour observer et analyser la dynamique spatiale en temps réel du fuseau mitotique chez la levure bourgeonnante. Les programmes développés permettent de localiser dans l'espace tridimensionnel des structures subcellulaires et de détecter leurs déplacements au cours du temps. Le marquage par protéines fluorescentes des pôles du fuseau mitotique et de la membrane cellulaire a permis de quantifier la dynamique du fuseau mitotique dans un système de coordonnées pertinent pour la division cellulaire. Je décrirai des simulations numériques de signaux fluorescents de ces structures subcellulaires qui m'ont permis de caractériser la fiabilité et de quantifier la précision des programmes d'analyse.
Je terminerai ce mémoire par la description d'un dispositif microfluidique permettant à la fois la culture de cellules et la caractérisation de leur dynamique à l'échelle individuelle, et ce sur de longues échelles de temps. Les outils développés au cours de cette thèse et présentés dans ce mémoire offrent la possibilité d'analyses quantitatives nouvelles des processus hors équilibre en jeu chez les cellules vivantes en général.
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Nixon, Faye Margaret. "A three-dimensional study of mitotic spindle ultrastructure." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3001861/.
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The mitotic spindle is the complex machinery that enables a cell to segregate its genetic material faithfully. Without fidelity in this process, genetic abnormalities may occur which, in turn, can lead to cancer. Microtubules of the mitotic spindle are bundled together to form kinetochore fibres which contribute to the movement of replicated chromosomes during cell division, helping to ensure the DNA is equally divided. Electron microscopy has long been used to observe inter-microtubule bridges within kinetochore fibres and, although they are thought to confer stability to the fibres thus ensuring faithful mitosis, their identity and function is yet to be fully established. These inter-microtubule cross-linkers were thought to be simple structures, providing a bridge directly between two microtubules. Here, three dimensional electron microscopy was used to examine these cross-linkers in unprecedented detail - revealing a complex network of microtubule connectors which we have termed 'the mesh'. To establish the functional role of the mesh, levels of a known microtubule crosslinker, the TACC3-chTOG-clathrin complex, were manipulated. In cells with too much mesh, kinetochore microtubules were found to be disorganised - forming tighter clusters within fibres and deviating from their parallel trajectories. Kinetochore fibres were also found to contain more microtubules than in control cells, and took longer to complete mitosis. These observations provide evidence that the mesh plays a functional role in the organisation within kinetochore fibres, directly impacting the ability of the cell to divide successfully. In addition to fine kinetochore fibre structure, scanning electron microscopy was optimised to examine changes to the whole spindle structure after manipulation of the mesh. This revealed changes to spindle architecture when microtubuleassociated proteins were altered, and offers insight into how cancer can be initiated when these proteins are dysregulated.
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SCARFONE, ILARIA GIUSY. "Spatial control of mitotic exit and spindle positioning in budding yeast." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/75407.
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The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e. the bud neck) and the division axis (i.e. the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial.
We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.
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Schlaitz, Anne-Lore. "Regulation of Mitotic Spindle Assembly in Caenorhabditis elegans Embryos." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1181247079528-57268.
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The mitotic spindle is a bipolar microtubule-based structure that mediates proper cell division by segregating the genetic material and by positioning the cytokinesis cleavage plane. Spindle assembly is a complex process, involving the modulation of microtubule dynamics, microtubule focusing at spindle poles and the formation of stable microtubule attachments to chromosomes. The cellular events leading to spindle formation are highly regulated, and mitotic kinases have been implicated in many aspects of this process. However, little is known about their counteracting phosphatases. A screen for genes required for early embryonic cell divisions in C. elegans identified rsa-1 (for regulator of spindle assembly 1), a putative Protein Phosphatase 2A (PP2A) regulatory subunit whose silencing causes defects in spindle formation. Upon rsa-1(RNAi), spindle poles collapse onto each other and microtubule amounts are strongly reduced. My thesis work demonstrates that RSA-1 indeed functions as a PP2A regulatory subunit. RSA-1 associates with the PP2A enzyme and recruits it to centrosomes. The centrosome binding of PP2A furthermore requires the new protein RSA-2 as well as the core centrosomal protein SPD-5 and is based on a hierarchical protein-protein interaction pathway. When PP2A is lacking at centrosomes after rsa-1(RNAi), the centrosomal amounts of two critical mitotic effectors, the microtubule destabilizer KLP-7 and the kinetochore microtubule stabilizer TPXL-1, are altered. KLP-7 is increased, which may account for the reduction of microtubule outgrowth from centrosomes in rsa-1(RNAi) embryos. TPXL-1 is lost from centrosomes, which may explain why spindle poles collapse in the absence of RSA-1. TPXL-1 physically associates with RSA-1 and RSA-2, suggesting that it is a direct target of PP2A. In summary, this work defines the role of a novel PP2A complex in mitotic spindle assembly and suggests a model for how different microtubule re-organization steps might be coordinated during spindle formation.
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Smith, Christopher. "Kinetochore dynamics and their attachment to the mitotic spindle." Thesis, University of Warwick, 2016. http://wrap.warwick.ac.uk/83227/.
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Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle – a dynamic microtubule-based force-generating machine (Dumont & Mitchison, 2009). Connections between chromosomes and the spindle are mediated by kinetochores, pairs of multi-protein complexes assembled on centromeric chromatin that harness the pushing and pulling forces associated with microtubule dynamics to power chromosome movement (Rago & Cheeseman, 2013). This structure is reported to be compliant (Wan et al., 2009; Maresca & Salmon, 2009; Uchida et al., 2009; Dumont et al., 2012; Drpic et al., 2015; Etemad et al., 2015; Tauchman et al., 2015; Magidson et al., 2016), which is the basis upon which it can transduce forces, and satisfy mitotic checkpoints. However recent studies have suggested that the kinetochore is a non-compliant linkage (Suzuki et al., 2014), while others negate the requirement for intra-kinetochore stretch to allow onset of anaphase (Etemad et al., 2015; Tauchman et al., 2015; Magidson et al., 2016). It is therefore crucial that the reasons for these differences are elucidated, and a new model developed to explain regulatory pathways sensitive to intra-kinetochore structure. I have developed a novel semi-automated imaging assay to track kinetochore proteins tagged with two different coloured fluorescent proteins in 3D, coupled with a detailed assay for measurement of the required correction for 3D chromatic aberration directly from cells. I reveal using three dimensional tracking that the kinetochore forms a non-compliant linkage between centromeric chromatin and kintetochore spindle fibres. Instead the outer kinetochore layer can swivel around the inner kinetochore/centromere, which results in a reduction in intra-kinetochore distance when viewed in lower dimensions, the only 3D measurement thus far being by Etemad et al. (2015). I show that swivel provides a mechanical flexibility that enables kinetochores at the periphery of the spindle to engage microtubules. Swivel reduces as cells approach anaphase, explaining previous suggestions that the kinetochore needs to be stretched upon bi-orientation prior to anaphase onset (Maresca & Salmon, 2009; Uchida et al., 2009). Reduction in swivel suggests there is an organisational change linked to checkpoint satisfaction, and/or changes required in kinetochore mechanochemistry before sister chromatid dysjunction. I show that Cdk1 inactivation is required to suppress kinetochore-microtubule dynamics in anaphase, and therefore may mediate required changes in kinetochore state. Finally, my work opens up the possibility to map the 3D architecture of the kinetochore, and between different domains within proteins and complexes. With such an architectural understanding, it will now be possible to investigate the mechanisms of organisational changes that occur during cycles of microtubule attachment and the regulatory processes of error-correction and SAC activation/silencing.
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Zadra, Ivan 1991. "Understanding the role of polyglutamylation in the mitotic spindle." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2022. http://hdl.handle.net/10803/673829.
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To ensure faithful segregation of the genetic material, cells build the mitotic spindle, a machinery made of dynamic microtubules (MTs) and MT associated proteins (MAPs). Chromosome bi-orientation is achieved through the attachment of the kinetochore pairs to MTs emanating from the two opposite spindle poles. Kinetochore-MT (KMT) dynamics are finely tuned within a narrow range to support the error correction mechanism and accomplish error-free anaphases.
This work shows that mitotic spindle MT polyglutamylation triggered by the TTLL11 enzyme ensures chromosome segregation fidelity in HeLa cells and zebrafish embryos. Consistently, bioinformatics analysis reveals TTLL11 downregulation in human cancer, and a negative correlation of its expression with aneuploidy. MT polyglutamylation imbalance in HeLa cells leads to an enhanced MT stability without affecting mitotic progression and the functionality of the spindle assembly checkpoint (SAC). Altogether, these results suggest that MT polyglutamylation provides a novel SAC-independent mechanism that ensures chromosome segregation fidelity.Per tal d’assegurar una segregació fidel del material genètic, les cèl·lules construeixen el fus mitòtic, una maquinària feta de microtúbuls (MTs) dinàmics i proteïnes associades als MTs. La orientació bipolar dels cromosomes s’aconsegueix a través de la unió dels dos parells de cinetocors amb els MTs que sorgeixen dels dos pols oposats del fus mitòtic. Les dinàmiques cinetocor-MT estan controlades finament en un petit rang per tal de permetre el mecanisme de correcció d’errors i aconseguir anafases sense defectes. Aquest estudi mostra que la poliglutamilació dels MT del fus mitòtic mitjançada per l’enzim TTLL11 assegura una segregació fidel dels cromosomes en cèl·lules HeLa i embrions de peix zebra. De la mateixa manera, un anàlisi bioinformàtic revela l’existència d’una regulació a la baixa de TTLL11 en mostres humanes de càncer, i una correlació negativa de la seva expressió amb aneuploïdia. El desequilibri de poliglutamilació dels MTs en cèl·lules HeLa condueix a un augment de l’estabilitat dels MTs, sense afectar la progressió mitòtica i la funcionalitat del punt de control de l’acoblament del fus mitòtic (SAC). En conjunt, aquests resultats suggereixen que la poliglutamilació dels MTs constitueix un nou mecanisme independent del SAC que assegura la segregació fidel dels cromosomes en la divisió cel·lular.
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Dunsch, Anja Katrin. "Control of the mitotic spindle by dynein light chain 1 complexes." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:b2fd5670-a035-42ca-aaef-78a30aeaa084.
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Robust control mechanisms ensure faithful inheritance of an intact genome through the processes of mitosis and cytokinesis. Different populations of the cytoplasmic dynein motor defined by specific dynein adaptor complexes are required for the formation of a stable bipolar mitotic spindle. This study analysed how different dynein subcomplexes contribute to spindle formation and orientation. Various dynein subpopulations were identified by mass spectrometry. I have shown that the dynein light chain 1 (DYNLL1) directly interacts with the kinetochore localised Astrin-Kinastrin complex as well as the spindle microtubule associated complex formed by CHICA and HMMR. I have characterised both complexes and identified unique functions in chromosome alignment and mitotic spindle orientation, respectively. I have found that Kinastrin (C15orf23) is the major Astrin-interacting protein in mitotic cells and is required for Astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Fixed cell microscopy revealed that cells over-expressing or depleted of Kinastrin mislocalise Astrin. Additionally, depletion of the Astrin-Kinastrin complex delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset (Dunsch et al., 2011). Using immunoprecipitation and microtubule binding assays, I have shown that CHICA and HMMR interact with one another, and target to the spindle by a microtubule-binding site in the amino-terminal region of HMMR. CHICA interacts with DYNLL1 by a series of conserved TQT motifs in the carboxy-terminal region. Depletion of DYNLL1, CHICA or HMMR causes a slight increase in mitotic index but has little effect on spindle formation or checkpoint function. Fixed and live cell microscopy reveal, however, that the asymmetric distribution of cor tical dynein is lost and the spindle in these cells fails to orient correctly in relation to the culture surface (Dunsch et al., 2012). These findings presented here suggest that the Astrin-Kinastrin complex is required for normal spindle architecture and chromosome alignment, and that per turbations of this pathway result in delayed mitosis and non-physiological separase activation, whereas HMMR and CHICA act as par t of a dynein-DYNLL1 complex with a specific function defining or controlling spindle orientation.
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Sen, Onur. "Phospho-regulation of the spindle assembly checkpoint." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/15873.
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Mitosis is a highly regulated process by which a cell duplicates and distributes its chromosomal DNA into two identical daughter cells equally. Equal distribution of the chromosomes is crucial for accurate propagation of genetic information. This is essential for maintaining viability and preventing genomic instability that can potentially lead to cancer. In order to avoid unequal distribution of chromosomes, cells employ a surveillance mechanism called the spindle assembly checkpoint (SAC). The SAC is an inhibitory signalling network, which delays segregation of chromosomes, until they have stably attached to spindle microtubules through their multi-protein platforms, known as kinetochores. The main target of the SAC is the anaphase promoter complex/ cyclosome (APC/C), an E3 ubiquitin ligase. Specifically APC/C and its activator Cdc20 are inhibited by the main effector of the SAC, called the mitotic checkpoint complex (MCC). The MCC consists of Cdc20, Mad2, Mad3 and Bub3 (except S. pombe) proteins, which are recruited to the unattached kinetochores to promote MCC assembly. Once the chromosomes stably attach to the spindle, the SAC is turned off, MCC disassembles, and APC/CCdc20 is released from the inhibition. Activated APC/CCdc20 then targets its two main substrates, securin and cyclin B, for proteasomal degradation, and thereby triggers anaphase onset and mitotic exit. SAC signalling involves many protein components, whose activities are essentially regulated by direct protein-protein interactions and/ or post-translational modifications. One of these major modifications is phosphorylation, which is mediated by the SAC kinases such as Aurora B, Mps1 and Bub1. A number of studies have characterised SAC related substrates of Aurora B and Mps1 kinases in several model organisms. On the other hand, Bub1 kinase activity has been thought to play a key role in chromosome bi-orientation and more of an auxiliary role in SAC activation. The aim of this study is to investigate the importance of Bub1 kinase activity for SAC response in fission yeast Schizosaccharomyces pombe (S. pombe). SAC activation assays, using various degrees of spindle perturbation, have demonstrated that Bub1 kinase activity plays an important role in SAC maintenance. In order to examine the pathways downstream of Bub1, we set out to indicate Bub1 substrates which may be involved in SAC signalling. According to studies in various species, Cdc20 appears to be a prominent candidate, whose phosphorylation by Cdk1 and Bub1 kinases has been reported to regulate its mitotic activity. To investigate whether Cdc20 is phosphorylated by Bub1 in vitro, we purified recombinant S. pombe proteins from insect cells. Subsequent kinase assays identified Cdc20 as an in vitro substrate of Bub1, and the phosphorylated sites in Cdc20 were mapped by mass spectrometry. To address if this phospho-modification is involved in SAC regulation, phosphorylation mutants of Cdc20 were analysed in terms of their abilities to activate and silence SAC in vivo. Results show that phosphorylation of Cdc20 C-terminus promotes SAC maintenance in response to spindle damage. Furthermore, the mutations mimicking Bub1-mediated phosphorylation of Cdc20 Cterminus restore the SAC defects in the absence of Bub1 kinase activity. In addition, we purified S. pombe mitotic checkpoint complex (MCC) from insect cells, and analysed the interactions between its components (Cdc20, Mad2 and Mad3) by cross-linking mass spectrometry. Crystal structure of S.pombe MCC has been determined recently, which lacks Mad3 C-terminus and flexible C-terminal tail of Cdc20. Using an MCC with full length Mad3, we identified novel interactions between the C-terminal tails Mad3 and Cdc20, which are in close proximity to the identified Cdc20 phosphorylation sites. Briefly, in this study we confirm the previously known roles of Bub1 kinase activity (chromosome bi-orientation). Moreover, we propose a new pathway (in addition to the well-established H2A pathway) mediated by Cdc20, that may be important to maintain the SAC response.
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Campbell, Ian Winsten. "The Mitotic Exit Network detects spindle position and anaphase entry." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122062.
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This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The Mitotic Exit Network (MEN), an essential GTPase signal-transduction cascade, controls mitotic exit in budding yeast. The MEN protects genomic integrity by ensuring chromosome segregation is complete prior to cytokinesis. Two signals are required for MEN activation: (1) movement of the nucleus into the daughter cell and (2) anaphase onset. These two events only coincide after anaphase chromosome segregation, ensuring that mitosis is complete prior to cytokinesis. The MEN is regulated by spindle position. The MEN GTPase, Tem1, is inhibited as long as the entire spindle resides in the mother cell. Tem1 becomes active when spindle elongation along the mother-daughter axis drives half of the nucleus into the bud. If spindle elongation fails to move part of the nucleus into the daughter cell, MEN activation is prevented, providing time to reposition the spindle. In addition to this spatial regulation, activation of the MEN is restricted to anaphase by inhibitory cyclin-dependent kinase (Cdk) phosphorylation of the MEN kinase cascade. During anaphase onset, Cdk activity decreases; creating a temporal signal that releases the MEN from inhibition. This temporal signal prevents MEN activation should the nucleus move into the daughter cell prior to anaphase. By integrating multiple inputs the MEN creates a regulated cell-cycle transition that is responsive to cell-cycle stage and spindle position.
by Ian Winsten Campbell.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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GALATI, ELENA. "Yeast response to prolonged activation of the spindle assembly checkpoint." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/19557.
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Faithful chromosome segregation during mitosis is fundamental for cell viability and genome stability. For a correct division, all kinetochores must be attached to the mitotic spindle and cohesion must be timely removed. Anaphase is triggered by the Anaphase Promoting Complex bound to its regulatory subunit Cdc20 (APC-Cdc20) that polyubiquitylates securin (Pds1 in budding yeast), whose role is to maintain inactive the protease separase (Esp1 in budding yeast) until anaphase onset. Once active, separase cleaves cohesin, thus triggering sister chromatid separation. Separase also promotes cyclinB proteolysis and mitotic exit due to its involvement in the Cdc14-early anaphase release (FEAR) pathway that promotes a partial activation of the Cdc14 phophatase, which is in turn key for CDK inactivation and mitotic exit. Cdc14 is maintained inactive throughout most of the cell cycle bound to its inhibitor Net1/Cfi1 and trapped in the nucleolus. At the beginning of anaphase Cdc14 is released from the nucleolus into the nucleus by the FEAR pathway; subsequently, Cdc14 is released also in the cytoplasm by the MEN (Mitotic Exit Network) pathway. In this way Cdc14 is fully active and can trigger mitotic exit by cyclinB-CDK inactivation.
The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism conserved in all eukaryotic organisms that ensures the correct segregation of the genetic material. In fact, it inhibits the metaphase to anaphase transition until all kinetochores are properly attached to the mitotic spindle by inactivating the APC-Cdc20 complex, thus providing the time for error correction.
Cells do not arrest indefinitely upon SAC activation. After a variable period of time cells escape from the metaphase arrest also in the presence of a damaged mitotic spindle or faulty kinetochore attachments to spindle microtubules. This process is referred to as adaptation or mitotic slippage and is often involved in the resistance to chemotherapeutic compounds that target the mitotic spindle. In spite of its importance, the adaptation process is still little known.
Within this context, the goals of my Ph.D. were: (1) to characterize the molecular mechanisms underlying SAC adaptation and (2) to search for factors involved in this process. For these purposes we used the yeast Saccharomyces cerevisiae as a model organism.
(1) We characterized the adaptation process in either the presence or the absence of mitotic spindle perturbations. We depolymerized spindles by using two different drugs that alter microtubule dynamics, i.e. nocodazole and benomyl, whereas we induced SAC hyperactivation without spindle damage by overproducing Mad2 (GAL1-MAD2 cells), one of the key proteins for SAC signal generation and maintenance. We observed that in all the conditions cells are able to adapt, but with different kinetics. In particular, cells adapt faster in benomyl, while in nocodazole and with high levels of Mad2 cells need more time to slip out of mitosis. The few data available about SAC adaptation in higher eukaryotes indicate that SAC adaptation is accompanied by chromatid separation, a decrease in mitotic CDK activity and mitotic exit. Indeed, like in mammalian cells, yeast securin and cyclinB are degraded and sister chromatids are separated during adaptation. In addition, cyclinB stabilization, as well as Cdc20 and Cdc5 (polo kinase) inactivation, markedly delay adaptation, while the only yeast CKI (Sic1) is not involved in this process. Finally, when yeast cells adapt the SAC is likely to be turned off, as shown by the disassembly of the Mad1/Bub3 checkpoint complex.
(2) To search for factors involved in SAC adaptation, we performed a genetic screen using GAL1-MAD2 cells. In particular, we screened for mutants that would remain arrested for prolonged times in mitosis upon MAD2 overexpression. We identified Rsc2, a non-essential component of the RSC chromatin remodelling complex, as a regulator of SAC adaptation in yeast. We demonstrated that RSCRsc2 is involved in fine tuning mitotic exit during the unperturbed cell cycle. Its activity becomes particularly important in conditions that would activate the SAC, as it contributes to cyclinB degradation. In the absence of Rsc2 Net1 phosphorylation and the early anaphase release of Cdc14 from the nucleolus are impaired, whereas expression of a dominant allele of CDC14 that loosens Net1 inhibition (CDC14TAB6-1) is sufficient to restore mitotic exit in conditions where Rsc2 becomes essential for this process. We further demonstrated that the ATPase activity of RSC is required for mitotic exit regulation, suggesting that its chromatin-remodelling activity is involved in this process. By studying possible genetic interactions between the RSC2 deletion and FEAR or MEN mutations, we found that RSC2 deletion confers synthetic lethality or sickness to MEN but not to FEAR mutants. Altogether, our data suggest that RSCRsc2 is a novel component of the FEAR pathway. Finally, we demonstrated that Rsc2 interacts in vivo and in vitro with the polo kinase Cdc5, which controls mitotic exit at different levels.
Since RSC binds to acetylated histone tails, it is possible that histone transacetylases are also involved in SAC adaptation. We tested if the SAGA (Spt-Ada-Gcn5 Acetyltransferase) complex is involved in SAC adaptation by deleting ADA2 or GCN5 in yeast. Indeed, SAGA seems involved in adaptation, although the contribution of Ada2 and Gcn5 in the process differs depending on the conditions used to activate the SAC.
Finally, since we found that upon treatment with benomyl (a microtubule destabilizer) cells adapt dividing nuclei, we wondered if SAC adaptation could be linked to the presence of cytoplasmic microtubules that are still partially detectable in these conditions. We therefore asked whether motor proteins and microtubule regulators are involved in mitotic slippage. Indeed, we found that in the absence of Kip2 and Bik1, which specifically bind to cytoplasmic microtubules, cells divide nuclei and exit mitosis slower than wild type cells, demonstrating that cytoplasmic microtubules and associated proteins could accelerate SAC adaptation.
In conclusion, SAC adaptation is a very complex process whose timing probably depends on the interplay between different mechanisms. An important aim for a complete comprehension of this process, as well as for the development of new and more efficient cancer therapies, will be to identify novel factors implicated in adaptation and clarify how their function might be linked to one another.
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Hewitt, Laura. "Using a novel small molecule inhibitor to investigate the role of Mps1 kinase activity." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/using-a-novel-small-molecule-inhibitor-to-investigate-the-role-of-mps1-kinase-activity(fcacfefc-90d9-4e92-9af4-a57897737329).html.
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During mitosis, accurate chromosome segregation is essential: gain or loss of genetic information can be detrimental to cell viability, or promote tumourigenesis. The mitotic checkpoint (also known as the spindle assembly checkpoint or SAC) ensures accurate chromosome segregation by delaying cell cycle progression until accuracy can be guaranteed. Mps1 is a protein kinase that is crucial for mitotic checkpoint signalling and also for proper chromosome alignment at metaphase. However, the precise role of Mps1’s catalytic activity is still unclear. Here, I present AZ3146, a novel small molecule inhibitor of Mps1. AZ3146 inhibits recombinant Mps1 in vitro with an IC50 of ~35 nM, and has low activity against a panel of 50 kinases, suggesting a reasonable degree of selectivity. As predicted for an Mps1 inhibitor, AZ1346 treatment led to spindle checkpoint malfunction in cells, accelerated mitotic timing, and perturbed the kinetochore localisation of the checkpoint effector Mad2. AZ3146 has a negative effect on cell viability, suggesting it leads to detrimental missegregations. Thus, the cellular effects of AZ3146 are consistent with Mps1 inhibition, and I was able to use the compound confidently as a tool to further probe the role of Mps1 activity in cells.Strikingly, levels of Mps1 increased at unattached kinetochores following inhibition of its kinase activity, suggesting Mps1’s kinetochore localisation is regulated by its own activity. A kinase-dead GFP-Mps1 fusion protein only accumulated at kinetochores in the absence of endogenous, active Mps1, implicating intra-molecular interactions in regulation of Mps1’s kinetochore localisation. I confirm a role for Mps1 in the mechanism of chromosome alignment, but in contrast to previous reports I did not detect a decrease in Aurora B activity following Mps1 inhibition. On the contrary, both Mps1’s phosphorylation status and its kinetochore localisation were affected by treatment with the Aurora B inhibitor ZM447439, placing Mps1 downstream of Aurora B. As an alternative explanation for the alignment defect in cells with reduced Mps1 activity, I found that levels of the plus-end directed kinesin Cenp-E were markedly decreased at unaligned kinetochores. I propose a model in which catalytically active Mps1’s auto-release from kinetochores simultaneously promotes both mitotic checkpoint signalling and chromosome alignment by facilitating Mad2 dimerisation and Cenp-E binding at unattached kinetochores.
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Golub, Ognjen. "Molecular Mechanisms Regulating Subcellular Localization and Function of Mitotic Spindle Orientation Determinants." Thesis, University of Oregon, 2016. http://hdl.handle.net/1794/20711.
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Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle.
I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh).
I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent.
This dissertation includes unpublished and previously published co-authored material.10000-01-01
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Thein, Kerstin. "Functional characterization of the mitotic-spindle and kinetochore associated protein astrin." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-79583.
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Hüls, Daniela. "Structural and functional studies on mitotic spindle orientation in Saccharomyces cerevisiae." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-141524.
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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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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 bipolaireOnce 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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Nguyen, Thu Xuan Thi. "Genetic analysis of the amino terminus of spindle pole component spc110p /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5085.
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Nannas, Natalie Jo. "Investigation of Force, Kinetochores, and Tension in the Saccharomyces Cerevisiae Mitotic Spindle." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11047.
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Cells must faithfully segregate their chromosomes at division; errors in this process causes cells to inherit an incorrect number of chromosomes, a hallmark of birth defects and cancer. The machinery required to segregate chromosomes is called the spindle, a bipolar array of microtubules that attach to chromosomes through the kinetochore. Replicated chromosomes contain two sister chromatids whose kinetochores must attach to microtubules from opposite poles to ensure correct inheritance of chromosomes. The spindle checkpoint monitors the attachment to the spindle and prevents cell division until all chromatids are attached to opposite poles. Both the spindle and the checkpoint are critical for correct segregation, and we sought to understand the regulation of these two components. The spindle is assembled to a characteristic metaphase length, but it is unknown what determines this length. It has been proposed that spindle length could be regulated a balance of two forces: one generated by interaction between microtubules that elongates the spindle and a second due to interactions between kinetochores and microtubules that shortens the spindle. We tested this force-balance model which predicts that altering the number of kinetochores will alter spindle length. We manipulated the number of kinetochores and found that spindle length scales with the number of kinetochores; introducing extra kinetochores produces shorter spindles and inhibiting kinetochores produces longer spindles. Our results suggest that attachment of chromosomes to the spindle via kinetochores produces an inward force that opposes outward force. We also found that the number of microtubules in the spindle varied with the number of kinetochores. In addition to establishing a spindle, cells must also guarantee that chromosomes are correctly attached to it. Correct attachment generates tension as the chromatids are pulled toward opposite poles but held together by cohesin until anaphase. The spindle checkpoint monitors this tension which causes stretching of chromatin and kinetochores. Lack of tension on activates the checkpoint, but is unknown if the checkpoint measures stretch between kinetochores (inter-kinetochore stretch) or within kinetochores (intra-kinetochore). We tethered sister chromatids together to inhibit inter-kinetochore stretch and found that the checkpoint was not activated. Our results negate inter-kinetochore models and support intra-kinetochore models.
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Sundberg, Holly. "Analysis of the spindle pole component Spc110p /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/9225.
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Lu, Michelle. "The Construction and Deconstruction of Signaling Systems that Regulate Mitotic Spindle Positioning." Thesis, University of Oregon, 2013. http://hdl.handle.net/1794/12955.
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Signaling systems regulate the flow of cellular information by organizing proteins in space and time to coordinate a variety of cellular activities that are critical for the proper development, function, and maintenance of cells. Signaling molecules can exhibit several levels of complexity through the utilization of modular protein interactions, which can generate simple linear behaviors or complex behaviors such as ultrasensitivity. Protein modularity also serves as the basis for the vast protein networks that form the regulatory networks that govern several biological activities. My work focuses on the importance of protein modularity in complex biological systems, in particular the regulatory pathways of spindle positioning.
The first part of my work involves the construction of a synthetic regulatory network using modular protein interactions in an effort to understand the complex behavior of the natural spindle orientation regulator Pins. Utilizing well-characterized protein domains and their binding partners, I built an autoinhibited protein switch that can be activated by a small protein domain. We found that the input-output relationship of the synthetic protein switch could be tuned by the simple addition of "decoy" domains, domains that bind and sequester input signal, thereby impeding the onset of the output response to generate an input threshold. By varying the number and affinities of the decoy domains, we found that we could transform a simple linear response into a complex, ultrasensitive one. Thus, modular protein interactions can serve as a source of complex behaviors.
The second part of my work focuses on elucidating the molecular mechanisms underlying spindle positioning in the Drosophila neuroblast. I found that Pins orients the mitotic spindle by coordinating two opposite-polarity microtubule motors Dynein and Kinesin-73 through its multiple domains. Kinesin-73 also relies on its modular domain architecture to perform its duties in Pins-mediated spindle positioning, where its N-terminal half functions in coordinating cortical-microtubule capture while its C-terminal half functions as a region necessary for the activation of Dynein. Thus, modular protein design allows for the organization of spindle orientation regulators in space to achieve the complex biological activity that is spindle positioning.
This dissertation includes previously published and unpublished coauthored material.10000-01-01
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Garzon-Coral, Carlos. "The forces that center the mitotic spindle in the C. elegans embryo." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-163529.
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The precise positioning of the mitotic spindle to the cell center during mitosis is a fundamental process for chromosome segregation and the division plane definition. Despite its importance, the mechanism for spindle centering remains elusive. To study this mechanism, the dynamic of the microtubules was characterized at the bulk and at the cortex in the C. elegans embryo. Then, this dynamic was correlated to the centering forces of the spindle that were studied by applying calibrated magnetic forces via super-paramagnetic beads inserted into the cytoplasm of one- and two-cell C. elegans embryos. Finally, these results were confronted with the different centering models: cortical pushing model, cortical pulling model and the cytoplasmic pulling model.
This thesis shows that: (i) The microtubules dynamic of the spindle aster is controlled spatially in the C. elegans embryo, with not rescues and catastrophes in the cytoplasm but in the centrosome and the cortex, respectively. (ii) The centering mechanism of the spindle behaved roughly as a damped spring with a spring constant of 18 12 pN/ m and a drag coefficient of 127 65 pN s/ m (mean SD). This viscoelastic behavior is evidence of a centering force that recovers and/or maintains the position of the spindle in the cell center. (iii) It seems to be two mechanisms that recover/maintain the spindle position. A fast one that may work for transient displacements of the spindle and a slow one that work over large and long perturbations. (iv) The centering forces scale with the cell size. The centering forces are higher in the two-cell embryo. This result argues against a centering mechanism mediated by cytoplasmic factors. It seems to be a limit for the relation of centering force to size, as the forces found in the four-cell embryo are comparable to the single-cell ones. (v) The centering forces scale with the amount of microtubules in the cell. This strengthens the belief that the microtubules are the force transmission entities of the centering mechanism. (vi) The boundary conditions are important to maintain the centering forces. A transient residency time of microtubules at the cortex, which is controlled by cortical catastrophe factors, is indispensable for a proper force transmission by the microtubules. (vii) The elimination of cortical catastrophe factors provides evidence for microtubules buckling, which is taken as a proof of polymerization forces. (viii) The cortical pulling forces mediated by the gpr-1/2 pathway do not seem to be involved in centering and it is proposed they are present in the cell for off-center positioning purposes. (ix) The forces generated by vesicle transport are enough to displace the spindle and they are suggested to be auxiliary forces to centering. (x) The forces associated with the spindle change dramatically during cell division. From metaphase to anaphase the forces associated with the spindle scale up to five times. This behavior was consistent during the development of the embryo as the same pattern was observed in the one-, two- and four-cell embryo. (xi) The higher forces found during anaphase are not cortical pulling (via pgr-1/2 pathway) depended, and it is proposed the spindle is `immobilised' by tethering or by an unknown cortical pulling pathway.
To this date, this thesis presents the most complete in-vivo measurements of the centering forces in association with the microtubules dynamics. Taken together the results constrain molecular models of centering. This thesis concludes that most probably the predominant forces of the spindle centering mechanism during mitosis are generated by astral microtubules pushing against the cortex.
Additionally, this thesis presents the most complete map of forces during cell division during development, which will prove to be indispensable to understand the changes the spindle undergoes when it changes its function.
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Mazumdar, Aveek. "Mitotic spindle and centrosome defects induced by selective Plk1 inhibitor, Cyclapolin 1." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612097.
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Matos, Irina Alexandra Cardoso de. "Spatiotemporal regulation of mitotic spindle dynamics and its contribution for chromosome segregation." Doctoral thesis, Faculdade de Medicina da Universidade do Porto, 2009. http://hdl.handle.net/10216/55513.
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Garcia, Kristin Cruz. "Characterization of Drosophila Wee1 and its role in ensuring mitotic spindle fidelity." Connect to online resource, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3337094.
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Matos, Irina Alexandra Cardoso de. "Spatiotemporal regulation of mitotic spindle dynamics and its contribution for chromosome segregation." Tese, Faculdade de Medicina da Universidade do Porto, 2009. http://hdl.handle.net/10216/55513.
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Cotsiki, Marina. "Identification of novel protein interactors of the SV40 large T antigen using the yeast two hybrid system." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268375.
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Silkworth, William Thomas. "The effect of spindle geometry on the establishment of merotelic kinetochore attachment and chromosome mis-segregation." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/28241.
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At any given time there are on the order of one hundred million cells undergoing mitosis in the human body. To accurately segregate chromosomes, the cell forms the bipolar mitotic spindle, a molecular machine that distributes chromosomes equally to the daughter cells. To this end, microtubules of the mitotic spindle must appropriately attach the kinetochores: protein structures that form on each chromatid of each mitotic chromosome. The majority of the time correct kinetochore microtubule attachments are formed. However, mis-attachments can and do form. Mis-attachments that are not corrected before chromosome segregation can give rise to aneuploidy, an incorrect number of chromosomes. Aneuploidy occurring in the germ line can cause both miscarriage and genetic diseases. Furthermore, aneuploidy is a major characteristic of cancer cells, and aneuploid cancer cells frequently mis-segregate chromosomes at high rates, a phenotype termed chromosomal instability (CIN). CIN has been correlated with both advanced tumorigenesis and poor patient prognosis and over the years there have been many hypotheses for what causes CIN. In this study, we identified two distinct mechanisms that are responsible for CIN. Both of these mechanisms cause a transient, abnormal geometric arrangement of the mitotic spindle. Specifically, cancer cells possess supernumerary centrosomes, which lead to the assembly of multipolar spindles during early mitosis when attachments between kinetochores and microtubules are forming. Supernumerary centrosomes facilitate the formation of merotelic attachments, in which a single kinetochore binds microtubules from more than one centrosome. As mitosis progresses the supernumerary centrosomes cluster, giving rise to a bipolar spindle by the time of chromosome segregation. However, the high rates of merotelic attachments formed during the transient multipolar stage result in high rates of chromosome mis-segregation. The second geometric defect characterized is caused by failure of centrosomes to separate before kinetochore-microtubule attachments begin to form. This mechanism, too, leads to high rates of kinetochore mis-attachment formation and high rates of chromosome mis-segregation. Finally, this study shows that the mechanisms characterized here are prevalent in human cancer cells from multiple organ sites, thus revealing that both mechanisms are a common cause of CIN.Ph. D.
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Sze, Man-fong. "Characterization of mitotic checkpoint proteins, MAD1 and MAD2, in hepatocellular carcinoma." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36841286.
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Schatz, Christoph. "The role of the small GTPase ran during assembly of a mitotic spindle." [S.l.] : [s.n.], 2003. http://www.diss.fu-berlin.de/2003/262/index.html.
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Jaffrey, Ross G. "Genomic instability in cancer : the role of the mitotic spindle checkpoint gene hBUB1." Thesis, University of Aberdeen, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400744.
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In this study, a number of human cancer cell lines were characterised for their genomic instability phenotypes: using a panel of centromeric probes utilising the fluorescence in situ hybridisation (FISH) technique to detect CIN, and a microsatellite assay using radioactive PCR labelling to detect MIN. This characterisation allowed comparison of hBUB1 expression data, and assessed the differences between phenotypic cell line groups for hBUB1 mutations and polymorphisms. Having demonstrated the potential of radioactive-microsatellite PCR to display changes between drug sensitive and selected daughter drug resistant cell lines, steps to assess the actual hBUB1 2q region in primary resected human cancers, with corresponding normal tissue pairs, was taken. NSCLC and CRC tumours were used as models for evaluation of the hBUB1 locus. NSCLC and CRC subtypes were chosen due to the high incidence of CIN observed in these tumours. The hBUB1 locus on chromosome 2q was assessed for genomic instability using a panel of seven microsatellite markers. In addition, two novel CA dinucleotide repeats: BUBCA18 a 175bp CA20 product, and BUBCA19 a 119bp CA12 product each located within hBUB1 intronic sequence, were cloned and evaluated in the 32 CRC and 20 NSCLC samples. The microsatellite assessment demonstrated that the 2q locus was highly unstable in CRC, with 62.5% (n=20/32) patient sample pairs displaying MIN in one or more marker. NSCLC had no instability at any locus, suggesting that aneuploidy due to changes around the hBUB1 chromosome 2 locus was unlikely in this cancer subtype. The known hBUB1 mutations and selected polymorphisms were assessed in the two sets of patient groups, and in the human cancer cell lines characterised previously. Sequencing of the hBUB1 exons using flanking intronic primers and restriction fragment length polymorphism (RFLP) assays, developed for specific coding changes, were utilised to achieve this. No alterations were observed for the exon 4 and 13 mutations in all samples tested. Polymorphism analysis similarly revealed low frequencies in CRC and NSCLC. The novel hBUB1 microsatellite repeat, BUBCA18, displayed potential for the use as a clinical marker in detecting CRC within the general population. Results from our BUBCA18 pilot study demonstrated that heterozygosity for BUBCA18 was significantly associated with disease predisposition (OR 8.2 95%CI 28.24-2.68). This preliminary finding, and hypothesis that BUBCA18 could be used to test risk, was investigated further in a CRC case-control population: comprising 189 CRC cases and 251 matched controls. BUBCA18 profiles were assessed using the radioactive-PCR technique. The BUBCA18 case/control investigation showed that the initial hypothesis was incorrect, as heterozygosity of BUBCA18 was insignificant when used to predict CRC risk, within the larger sample group. The studies reported in this thesis have generated novel data regarding the impact of hBUB1 in human cancer. Initial evidence suggesting that hBUB1 has a significant role for CIN initiation seems unlikely.
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Lopes, Cláudia Sofia de Jesus. "Molecular partners for Bud6p-mediated orientation of the mitotic spindle in S. cerevisiae." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608848.
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Fink, Jenny. "Mechanotransduction in mitotic spindle positioning : Role of external forces and mechanical cortex properties." Paris 11, 2009. http://www.theses.fr/2009PA112117.
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L’orientation du fuseau mitotique est un processus clé qui est conservé des levures aux cellules animales. Il est essentiel pour la destinée cellulaire, pour le développement et pour l’organisation des tissues. Dans des cellules animales, il est généralement accepté que des stimuli externes polarisent le cortex d’actine, et que cette polarisation est ensuite transmise au fuseau mitotique et guide son positionnement. Au cours de ma thèse, j’ai étudié l’influence des forces extracellulaires sur l’orientation du fuseau mitotique dans des cellules mammifères en culture. Nous avons pu montrer que ces forces extracellulaires, transmises au corps cellulaire mitotique par l’intermédiaire des fibres de rétraction, étaient capables de diriger l’orientation du fuseau. Nous avons donc identifié une fonction nouvelle pour la mécano-transduction - la conversion de forces mécanique en signaux biochimiques qui finalement induisent une réponse cellulaire - pendant la mitose. Ces découvertes démontrent également que les signaux de polarisation biochimiques – qui ont été beaucoup étudiés par le passé – ne sont pas les seuls signaux régulant l’orientation du fuseau. Par ailleurs, nous avons pu montrer que le cortex d’actine était mécaniquement polarisé pendant la mitose: un quadrant du cortex était très souvent jusqu’à deux fois plus rigide que les trois autres quadrants. Le fuseau mitotique était aligné le long de ce gradient de rigidité avec un pole du fuseau apposé au quadrant le plus dur, un comportement qui a été également prédit par des simulations effectuées dans l’équipe de François NedelecIn mitosis, positioning of the microtubule spindle represents a key process that is conserved from yeast to animal cells. It is essential for cell fate, development and tissue organization and perturbations of this process can have as dramatic effects as uncontrolled cell dissemination and death of the whole organism. In animal cells, external stimuli are thought to polarize the actin cortex, and this polarization is subsequently transduced to the microtubule spindle leading to its positioning. During my thesis, I studied the influence of extracellular pulling forces on mitotic spindle orientation in cultured cells. We demonstrated that these extracellular forces that were transmitted to the mitotic cell body via retraction fibres could direct spindle positioning. We thus identified a novel function for mechanotransduction, i. E. The conversion of mechanical forces into biochemical signals that finally induce a cellular response, in the context of mitotic spindle positioning. These findings additionally demonstrate that biochemical cues -predominantly investigated by previous studies - are not the only important signals regulating spindle positioning. We could furthermore show that the actin cortex is mechanically polarized during mitosis: one cortex quadrant was often up to twice stiffer than the remaining three quadrants. The mitotic spindle appeared to be aligned with this stiffness gradient, one pole facing the stiffest quadrant. Simulations of spindle dynamics, performed in the group of François Nedelec, could predict this observed behaviour when using our measured parameters for cortical rigidity and microtubule dynamics
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Pietruszka, Patrycja. "Role of Tem1 phosphorylation in the control of mitotic exit and spindle positioning." Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON1T021.
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Dans la levure S. cerevisiae, la mitose nécessite le positionnement du fuseau mitotique le long de l’axe cellule mère-bourgeon (future cellule fille) afin d‘assurer une bonne ségrégation des chromosomes. Ce phénomène requiert le fonctionnement de deux mécanismes impliquant les protéines Kar9 et Dyn1. Durant la métaphase, Kar9 se positionne de manière asymétrique le long du fuseau mitotique, avec une accumulation notable sur les microtubules qui émanent de l’ancien « spindle pole body » (SPB; l’équivalent du centrosome dans les vertébrés), qui est normalement dirigé vers le bourgeon. Dans le cas d’un défaut d’alignement du fuseau mitotique, un mécanisme appelé « Spindle Position Checkpoint » (SPOC) inhibe la sortie de mitose et la cytokinèse, afin de permettre un réalignement correct du fuseau mitotique. La principale cible de ce checkpoint est une GTPase Tem1. Dans le cas d’alignement correct du fuseau mitotique, Tem1 active une voie de signalisation appelée le « Mitotic Exit Network » (MEN) qui permet de mener à la sortie de mitose et à la cytokinèse. Lors de la transition métaphase/anaphase Tem1 se positionne asymétriquement sur les SPBs jusqu’à se concentrer majoritairement sur l’ancien SPB. Des données récentes ont montré que des composants du MEN, Tem1 inclus, sont également impliqués dans la régulation de la localisation de la protéine Kar9 à l’SPB, et dans l’établissement d’une polarité correcte des SPBs durant la métaphase. En effet, Kar9 se positionne plus symétriquement dans le cas des mutants du MEN que dans le type sauvage, ce qui engendre des problèmes d’orientation du fuseau et de ségrégation des SPBs. Nous cherchons à élucider comment l’activité du MEN régule la localisation de Kar9 et l’orientation du fuseau mitotique en métaphase alors que les fonctions du MEN liées à la sortie de mitose restent bloquées jusqu’à la télophase. Nous avons émis l’hypothèse que les modifications post-traductionnelles de Tem1 pourraient jouer un rôle dans la régulation du MEN. Il a été montré que les résidus Y40 et Y45 sont phosphoryles in vivo. Afin de disséquer le rôle de ces résidus nous les avons mutés en phénylalanines. Ces mutations peuvent complémenter la létalité induite par la délétion de TEM1, suggérant que ce mutant conserve les fonctions essentielles de Tem1. Par ailleurs, la cinétique de progression du cycle cellulaire du mutant est la même que celle du type sauvage, signifiant que la perte de phosphorylation sur Tem1 ne semble pas agir sur la sortie de mitose. De plus, l’allèle mutant n’affecte pas la localisation aux SPBs de Tem1 ni celle de sa « GTPase-activating protein » Bub2/Bfa1 durant le cycle cellulaire. Bien que l’activité GTPasique de la protéine Tem1-Y40F,Y45F soit réduite in vitro, les mutations ne causent pas des défauts de SPOC in vivo et le mutant répond efficacement au mauvais alignement de fuseau mitotique en s’arrêtant en anaphase. Tous ces résultats nous suggèrent que la perte de phosphorylation de Tem1 n’affecte pas les fonctions de fin de mitose de cette GTPase. Par contre, nous avons découvert que la phosphorylation de Tem1 est requise pour la localisation asymétrique de Kar9 sur les SPBs, ainsi que pour l’alignement correct du fuseau mitotique durant la métaphase (la distribution de Kar9 est plus symétrique dans les cellules TEM1-Y40F,Y45F et que le fuseau mitotique n’est pas aligné correctement). Nous cherchons alors à trouver quelle kinase phosphoryle Tem1 et régule son activité. Les kinases potentielles sont la protéine Swe1 (la seule vraie kinase phosphorylant les tyrosines dans la levure) ainsi que la kinase Mps1 (kinase qui contrôle la duplication des SPBs). Nous développons actuellement des outils nous permettant de vérifier l’implication de ces deux candidats. Mots clés : Tem1, Kar9, cycle cellulaire, Mitotic Exit Network (MEN), Spindle Position Checkpoint (SPOC), phosphorylation on tyrosinesIn the budding yeast Saccharomyces cerevisiae a faithful mitosis requires positioning of the mitotic spindle along the mother-bud axis to ensure proper chromosome segregation. This is achieved by two distinct but functionally redundant mechanisms that require the APC (adenomatous polyposis coli)-like protein Kar9 and dynein (Dyn1), respectively. During metaphase, Kar9 localizes asymmetrically on the mitotic spindle, with a prominent accumulation on astral microtubules emanating from the old spindle pole body (SPB – i.e. the yeast equivalent of the centrosome) that is normally directed towards the bud. In case of spindle misalignment, a surveillance mechanism called Spindle Position Checkpoint (SPOC) inhibits mitotic exit and cytokinesis, thereby providing the time necessary to correct spindle alignment. The main target of the SPOC is the small GTPase Tem1, which activates a signal transduction cascade called Mitotic Exit Network (MEN) that drives cells out of mitosis and triggers cytokinesis. Tem1 is localized at SPBs, with an increasingly asymmetric pattern during the progression from metaphase to anaphase, when Tem1 is concentrated on bud-directed old SPB. Recent data have implicated MEN components also in the regulation of Kar9 localization at SPBs and in setting the right polarity of SPBs inheritance during metaphase. In particular, Kar9 localizes more symmetrically in MEN mutants than in wild type cells and this leads to spindle orientation and SPB inheritance defects (i.e. with the new SPB being oriented towards the bud). A key question emerging from these data is how MEN activity is regulated to promote proper Kar9 localization and spindle positioning in metaphase, while being restrained until telophase for what concerns its mitotic exit and cytokinetic functions. We hypothesised that Tem1 post-translational modifications might be relevant for this control and for this reason we have been focusing on the role of Tem1 phosphorylation. Tem1 was found in a wide phosphoproteomic study to be phosphorylated on two tyrosines (Y40 and Y45) located at its N-terminus. We constructed a non-phosphorylatable mutant, TEM1-Y40F,Y45F, where the two phosphorylated tyrosines were mutated to phenylalanine. This mutant allele was able to rescue the lethality caused by TEM1 deletion, suggesting that it retains all its the essential functions. The kinetics of cell cycle progression of TEM1-Y40F,Y45F cells was similar to that of wild type cells, suggesting that lack of Tem1 phosphorylation is unlikely to affect mitotic exit. In addition, the TEM1-Y40F,Y45F allele did not affect the SPB localization of Tem1 and its regulatory GTPase-activating protein Bub2/Bfa1 during the cell cycle. Moreover, although the Tem1-Y40F,Y45F mutant protein showed reduced GTPase activity in vitro, it did not cause SPOC defects in vivo and could efficiently respond to spindle mispositioning. Altogether, these results suggest that lack of Tem1 phosphorylation does not affect the late mitotic functions of the GTPase. In contrast, we found that Tem1 phosphorylation is required for Kar9 asymmetry at SPBs and proper spindle positioning during metaphase. Indeed, TEM1-Y40F,Y45F cells display a more symmetric pattern of Kar9 distribution at SPBs in this cell cycle stage, as well as spindle position and orientation defects. We are currently investigating if Tem1 phosphorylation also regulates the pattern of SPB inheritance. Finally, an important question that we are trying to answer is “what is the kinase that phosphorylates Tem1?” The best candidates are the wee1-like kinase Swe1, which is the only true tyrosine kinase of budding yeast, and Mps1, a dual-specificity protein kinase controlling SPB duplication. While we are developing specific tools to study Tem1 phosphorylation and ultimately identify its promoting kinase, we gained preliminary data suggesting that both kinases might be involved in spindle positioning
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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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Neurohr, Gabriel Erich. "A midzone-based ruler adjusts chromosome compaction to analphase spindle length." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/81932.
Full textAbstract:
Partitioning of chromatids during mitosis requires that chromosome
compaction and spindle length scale appropriately with each other. However,
it is not clear whether chromosome condensation and spindle elongation are
linked. Here we have used chromosome fusions to examine the impact of
increased chromosome length during yeast mitosis. We find that yeast cells
could cope with a >50% increase in the length of their longest chromosome
arm by decreasing the physical length of the mitotic chromosome arm through
1) reducing the number of copies of the repetitive rDNA array and 2) by
increasing the level of mitotic condensation. Consistently, cells carrying the
fused chromosomes became more sensitive to loss of condensin- and its
regulator polo kinase/Cdc5. Length-dependent stimulation of condensation
took place during anaphase and depended on aurora/Ipl1 activity, its
localization to the spindle midzone, and phosphorylation of histone H3 on
Ser10, a known Ipl1 substrate. The anaphase spindle therefore may function
as a ruler to adapt the condensation of chromosomes to spindle length.
Consistent with this, chromosome condensation levels correlate with the
length of anaphase spindles.
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Yoder, Tennessee Joplin. "The spindle pole body in Saccharomyces cerevisiae is a dynamic structure /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/5014.
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Chan, Ying Wai. "Induction of mitotic cell death and cell cycle arrest by spindle disruption and premature entry into mitosis after DNA damage /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BICH%202007%20CHANY.
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Vodicska, Barbara [Verfasser], and Ingrid [Akademischer Betreuer] Hoffmann. "Deciphering the function of MISP in mitotic spindle orientation / Barbara Vodicska ; Betreuer: Ingrid Hoffmann." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/117704370X/34.
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Li, Deyu. "Spatial and temporal regulation of mitotic progression by the spindle checkpoint in Drosophila melanogaster." Thesis, University of Newcastle Upon Tyne, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491836.
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The spindle assembly checkpoint is a cell-cycle surveillance system which ensures the mother cell equally and faithfully separates its replicated DNA contents into two daughter cells before the onset of anaphase. Mad2, BubRl and Cdc20 (Fzy in Drosophila melanogaster) are all kinetochore proteins which dynamically turnover on and off the mitotic kinetochores. It is believed that these proteins are forming a diffusible kinetochore anaphase inhibitory signal for spindle checkpoint function, inhibiting APC/C activity in order to delay the metaphase-anaphase transition (Musacchio and Salmon, 2007). APC/C is an E3 ubiquitin ligase. It ubiquitinates numbers of cell cycle regulating proteins, such as Cyclin B and Securin, and allows them to be targeted by proteasomes for destruction (Peters, 2006). However, the mechanism of the spatial and temporal interaction of these checkpoint proteins (Mad2, BubRl and Cdc20/Fzy) on kinetochores, and how they assemble to form this inhibitory signal remains unclear.
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Lock, Rowena Lesley. "Investigation into how mitotic spindle checkpoint function is compromised by Sv40 large T antigen." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444978/.
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The viral oncoprotein Simian virus 40 large T antigen (LT) efficiently immortalises primary rodent cells and occasionally transforms them to tumourigenicity. It has long been known that LT can cause genomic instability and induce aneuploidy, and that it disrupts the mitotic spindle checkpoint, involved in monitoring the segregation of sister chromatids. LT has recently been shown to interact with the spindle checkpoint protein Bubl. This thesis describes research into the effects of LT on spindle checkpoint function and examines the possibility that interaction of LT with Bubl is responsible. Novel monoclonal antibodies against Bubl were developed to aid this research. The interaction site of Bubl was mapped to amino acids 89-97 of LT using co-immunoprecipitation experiments in several cell lines, each expressing LT with a different mutation in the amino-terminal region. The non-Bub 1-binding (dl89-97) mutant was capable of immortalising rat embryo fibroblasts as efficiently as wild- type LT, but was defective for focus formation, perhaps suggesting a role for Bubl in the mechanism for transformation by LT. In the presence of the microtubule-depolymerising drug nocodazole, the spindle checkpoint is activated and cells arrest in mitosis. LT was shown to compromise spindle checkpoint function, allowing some cells to pass through to the next cell cycle even in the presence of nocodazole. However, without Bubl interaction, LT loses this ability and the spindle checkpoint is robust. An investigation of the checkpoint proteins and complexes present during spindle checkpoint activation in cells expressing wild-type or dl89-97 LT was initiated to analyse possible mechanisms for perturbation of the spindle checkpoint by LT. This study raises the possibility that LT induces genetic instability in mammalian cells by perturbation of spindle checkpoint function, and that this may be a critical component of the mechanism by which LT transforms cells.
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Fernández, Baldovinos Javier [Verfasser], and Thomas [Akademischer Betreuer] Worzfeld. "Mechanisms of Mitotic Spindle Orientation by Plexin-B2 / Javier Fernández Baldovinos ; Betreuer: Thomas Worzfeld." Marburg : Philipps-Universität Marburg, 2021. http://d-nb.info/1228535744/34.
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Duncan, Tommy. "Investigating the function of Drosophila MAPs Msd1 and dTD-60 in mitotic spindle assembly." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:3ed4021f-2ccc-4821-b7c5-40b06d5639b7.
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Mitosis is the process by which new cells are formed. Following accurate duplication of chromosomes, a cell must segregate its chromosomes into separate daughter cells with great accuracy. Failure to do so can result in genomic instability. Thus, entry into mitosis is tightly regulated via complex protein interactions. Microtubules (MTs) are versatile Tubulin polymers that constitute a considerable portion of the cytoskeleton, and it is the dramatic rearrangement of MTs upon mitotic entry that is required to build the mitotic spindle – the structure responsible for segregating the duplicated sister chromatids. MTs are modulated by MT-Associated Proteins (MAPs) that enact major MT rearrangements during mitosis. Identifying and understanding the role of MAPs is essential to the study of MT behaviour during mitosis. Recently, an RNAi screen for Drosophila MAPs identified two proteins that are the subject of this thesis; Msd1 and dTD-60. This thesis demonstrates that Msd1 is a novel MAP that is a component of the recently identified Augmin complex – the action of which is to generate a novel and redundant population of MTs within the mitotic spindle from existing MTs. Experiments described below demonstrate that Msd1 is required for Augmin-directed MT generation, and further investigate the role of this novel population of MTs within the developing fruit fly. Furthermore, a role for Msd1 in central spindle formation during anaphase in Drosophila is identified. dTD-60, the Drosophila homologue of human TD-60 (hTD-60), is the subject of another study described in this thesis. While hTD-60 has a role in metaphase progression through interaction with the Chromosomal Passenger Complex, a contrasting role for dTD-60 is investigated here. This thesis describes both a divergent localisation and phenotype of dTD-60, and further investigates the role of dTD-60 and its interactors in mitotic spindle formation.
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Orr, Bernard Nunes de Almeida. "The role of Mad2 and BudR1 in the spindle assembly checkpoint and mitotic progression." Doctoral thesis, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/50174.
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Hueschen, Christina Lynn. "Building the Mitotic Spindle| Spatial Regulation and Function of Force at Microtubule Minus-Ends." Thesis, University of California, San Francisco, 2019. http://pqdtopen.proquest.com/#viewpdf?dispub=13423508.
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Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped shape despite microtubule turnover and internal stresses. This ellipsoidal architecture, in which microtubule minus-ends are focused into two poles, is essential to the spindle’s function of accurately segregating chromosomes. In this work, I ask how the spindle forms and holds its steady-state shape. I report that the molecular motor dynein and the microtubule binding-protein NuMA are essential for mammalian spindles to reach and hold a steady-state geometry. In their absence, the kinesin-5 Eg5 powers a turbulent microtubule network that can drive flow of cytoplasmic organelles and whole-cell movement. Dynein and NuMA were previously known to be essential for spindle pole formation, but we did not know their contribution to shape stabilization at the whole-spindle scale—nor did we know how and where they pull on microtubules to build poles. Using quantitative live imaging and laser ablation, I show that dynein pulls specifically on microtubule minus-ends, rapidly transporting them towards poles. Dynein localization to microtubule minus- ends depends on NuMA, which recruits the dynein adaptor dynactin to minus-ends. Contrary to previous models, NuMA localization to minus-ends is independent of dynein and involves a C-terminal region outside its canonical microtubule-binding domain. Thus, NuMA serves as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles. This microtubule end-clustering compacts the spindle microtubule network to a defined geometry and suppresses network turbulence, maintaining a steady-state spindle shape over long timescales.
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Orr, Bernard Nunes de Almeida. "The role of Mad2 and BudR1 in the spindle assembly checkpoint and mitotic progression." Tese, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/50174.
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Tipton, Aaron R. "How to Assemble a Functional Mitotic Checkpoint Complex." University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1341597834.
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Sze, Man-fong, and 施敏芳. "Characterization of mitotic checkpoint proteins, MAD1 and MAD2, in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B38438550.
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Florian, Stefan [Verfasser]. "Bipolar spindle formation beyond Eg5: A study on antagonists and synergists of the molecular motor Eg5 during mitotic spindle formation / Stefan Florian." Konstanz : Bibliothek der Universität Konstanz, 2011. http://d-nb.info/1038383951/34.
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Leontiou, Ioanna. "'SynCheck' : new tools for dissecting Bub1 checkpoint functions." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33246.
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The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down's syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or 'Syncheck' is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the "wait anaphase" signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions.