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Inactivation of a human kinetochore by specific targeting of chromatin modifiersCardinale, Stefano January 2008 (has links)
Here I describe the construction and characterization of a new generation of human artificial chromosome that contains an array of DNA sequences that can be used to manipulate the chromosome in vivo and possibly in vitro. This HAC was originated in human fibrosarcoma HT1080 cells from a synthetic alphoid DNA containing an array of TetOperator sequences, cloned in a BAC-based vector. This synthetic ά-satellite DNA formed HACs that were stably maintained throughtout replication and segregation in HT1080 cells. However, I succeeded to also transfer and manipulate the alphoidtetO HAC into a HeLa-based hybrid cell line. The synthetic alphoidtetO HAC chromatin was similar to the chromatin at endogenous centromeric alphoid DNA. Importantly, the DNA sequences embedded in the synthetic HAC were accessible to targeting TetR-fused constructs in vivo. The alphoidtetO HAC could be successfully targeted with a number of TetR:fusion proteins without affecting its chromatin structure, kinetochore assembly and mitotic behaviour. However, the targeting of a transcriptional activator (tTA) inactivated the HAC synthetic alphoidtetO DNA in a fraction of transfected cells. Surprisingly, the targeting of the transcriptional repressor tTS, co-repressor KAP1 or the heterochromatin-associated protein HPIά severely inactivated the synthetic alphoidtetO kinetochore . In fact, upon targeting several inner and outer kinetochore proteins were delocalized from the alphoidtetO sequences. The dissociation of kinetochore proteins CENP-H and CENP-C appeared to precede that of CENP-A. The alphoidtetO HAC lacking inner kinetochore protein complexes showed mitotic defects including misalignment at the metaphase plate and defective anaphase segregation, ultimately being included in tiny DAPI-positive nano-nuclei in the cytoplasm. The transcriptional repressor tTS repressed the low levels of transcription from the alphoidtetO sequences. In addition, targeting of transcriptional repressors altered the HAC chromatin towards a more “closed”, heterochromatic conformation, as seen from the changes in histone tail modifications. Interestingly, the targeting of the histone methyltransferase EZH2 to the alphoidteto HAC showed a much milder inactivating activity compared to KAP1. Based on these results, I propose that the formation of HPI-type of heterochromatin or accumulation of HPIά to the centromeric regions could disrupt the association of constitutive kinetochore proteins to the underlying sequences. Centromeric alphoid sequences lacking a functional kinetochore structure then also loose the centromere-specific histone H3 variant CENP-A becoming definitively inactive. Alternatively, a basal transcriptional activity from centromeric sequences might be required for centromere functionality.
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Analysis of Mph1 kinase and its substrates in spindle checkpoint signallingZich, Judith January 2010 (has links)
Accurate chromosome segregation is crucial as mis-segregation results in aneuploidy, which can lead to severe diseases such as cancer. The spindle checkpoint monitors sister-chromatid attachment and inhibits the onset of anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle. The spindle checkpoint machinery of S.pombe is composed of many proteins, one of which is the kinase Mph1 (Mps1p-like pombe homolog). It previously has been shown that Mph1 is essential for the spindle checkpoint but not whether this is due to its kinase activity. In this study we determined the role of Mph1 kinase activity in the spindle checkpoint. To do so a kinase-dead version of Mph1, which had no detectable kinase activity, was analysed. Using this kinase-dead allele we showed that lack of Mph1 kinase activity abolished the spindle checkpoint and led to chromosome missegregation. As a result of these two defects cell viability of cells lacking Mph1 kinase activity was severely impaired. These results led to the question of how Mph1 kinase activity regulates the spindle checkpoint. Spindle checkpoint signalling is thought to mainly take place at two sites, at the kinetochore and at the anaphase promoting complex (APC). The APC is an E3 ubiquitin ligase that drives cells into anaphase by targeting the separase inhibitor securin and cyclin B for degradation by the 26 S proteasome. Upon activation of the spindle checkpoint the APC is inhibited by the mitotic checkpoint complex (MCC) composed of Slp1, Mad2 and Mad3. In this study we wanted to test whether the regulatory role of Mph1 kinase in the spindle checkpoint is via MCC binding to the APC. Using the kinase-dead version of Mph1 we showed that Mad2 and Mad3 binding to the APC is severely impaired in the absence of Mph1 kinase activity. This result led to the hypothesis that Mph1 might regulate Mad2 and Mad3 binding Using kinase assays Mad2 and Mad3 were identified as in vitro substrates of Mph1 and phosphorylation sites in Mad2 and Mad3 were determined by mass spectrometry. Phosphorylation mutants of Mad2 and Mad3 showed spindle checkpoint defects, indicating that they are important Mph1 substrates.
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Studies in oocytes from three mammalian species demonstrate that meiotic kinetochores are composed of previously unidentified subdomains and reveal two novel mechanisms behind the maternal-age effect in humansZielinska, Agata Pamela January 2019 (has links)
Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.
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Deciphering the Role of Aft1p in Chromosome StabilityHamza, Akil 25 January 2012 (has links)
The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.
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Deciphering the Role of Aft1p in Chromosome StabilityHamza, Akil 25 January 2012 (has links)
The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.
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Deciphering the Role of Aft1p in Chromosome StabilityHamza, Akil 25 January 2012 (has links)
The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.
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Étude de partenaires protéiques d’une protéine associée aux microtubules, MAP65-3, indispensable à la formation des cellules géantes induites par le nématode à galles Meloidogyne incognita : caractérisation du complexe de surveillance de la mitose chez Arabidopsis / Non disponiblePaganelli, Laëtitia 11 June 2013 (has links)
Les nématodes à galles du genre Meloidogyne sont des parasites obligatoires des plantes. Lors de l’interaction compatible, ils induisent la formation de cellules nourricières hypertrophiées et plurinucléées leur permettant d’assurer croissance et reproduction. L'étude des mécanismes moléculaires impliqués dans la formation de ces cellules géantes a permis d’identifier une protéine associée aux microtubules, MAP65-3, essentielle à la formation de ces cellules géantes et au développement du nématode. Un des partenaires protéiques de MAP65-3 est un homologue de BUB3, membre du « Mitotic Checkpoint Complex » (MCC). Le MCC est un point de contrôle de la mitose assurant la fidélité de la ségrégation des chromosomes. Au cours de ma thèse, j'ai caractérisé chez la plante modèle Arabidopsis thaliana les homologues du MCC: BUB3.1, MAD2 et la famille multigénique composée de BUBR1, BRK1 et BUB1.2. J’ai démontré les interactions in planta entre les membres du complexe, certaines interactions ayant lieu au niveau des noyaux, voire au niveau des centromères. J’ai réalisé l’analyse fonctionnelle de ces gènes et montré qu’ils étaient exprimés dans les tissus enrichis en cellules en division comme MAP65-3. L’étude de la localisation subcellulaire des protéines a révélé une localisation cytoplasmique pour BUB3.1, BUB1.2 et MAD2, nucléaire pour BUBR1 et centromérique pour BRK1. Nous avons pu également montrer que lorsque des défauts d’attachement des microtubules du fuseau mitotique sont provoqués, BUB3.1, BUBR1 et MAD2 se relocalisent au niveau des kinétochores. L’étude de la famille BUB1/BUBR1 a révélé que l’inactivation des gènes correspondants induisait une sensibilité accrue à un traitement chimique déstabilisant les réseaux de microtubules. L’étude de la mitose chez ces mutants a révélé que BUBR1 est essentielle à la réalisation d’une mitose sans erreur chez Arabidopsis. Ce travail a ainsi permis de caractériser pour la première fois le MCC chez A. thaliana. / Root-knot nematodes from the genus Meloidogyne are obligate biotrophic plant parasites. During a compatible interaction, they induce the redifferentiation of root cells into multinucleated and hypertrophied feeding cells to ensure their growth and reproduction. The study of molecular and cellular mechanisms underlying giant cell ontogenesis has led to the identification of a Microtubule-Associated Protein, MAP65-3, essential for giant cell ontogenesis and nematode development. One of the MAP65-3 interacting partners is a BUB3 homologue, member of the Mitotic Checkpoint Complex (MCC). The MCC is a surveillance mechanism ensuring that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the mitotic spindle. During my thesis, I have characterized the Arabidopsis thaliana orthologs of the MCC, BUB3.1, MAD2 and the multigenic family composed of BUBR1, BRK1 et BUB1.2. I have demonstrated that MAP65-3 and all the MCC members interact together in planta, some interactions taking place within the nuclei or at the centromeres. As MAP65-3, all these genes are expressed in dividing cells. The study of the subcellular localization of the protein showed a cytoplasmic localization for BUB3.1, BUB1.2 and MAD2, nuclear for BUBR1 and centromeric for BRK1. Thus, the MCC proteins did not relocalize to the kinetochore during a normal mitosis in planta. BUB3.1, BUBR1 and MAD2 localize to the unattached kinetochores following defects in spindle assembly as observed in cells treated with microtubule poisons. The functional analysis of BUB1/BUBR1 multigenic family showed that the knock-out mutants were more sensitive to microtubule-destabilizing drugs. Furthermore, analysis of mitosis revealed that BUBR1 is essential for an error-free mitosis in Arabidopsis. This work represents the first characterization of the MCC in A. thaliana.
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Deciphering the Role of Aft1p in Chromosome StabilityHamza, Akil January 2012 (has links)
The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.
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Examining the Regulation and Functions of Centrosomal Mps1Marquardt, Joseph R. 11 August 2017 (has links)
No description available.
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The effect of spindle geometry on the establishment of merotelic kinetochore attachment and chromosome mis-segregationSilkworth, William Thomas 27 July 2012 (has links)
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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