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.

The 2 micron plasmid of Saccharomyces cerevisiae is a multi-copy circular DNA genome that resides in the nucleus and exhibits nearly chromosome-like stability in host populations. Several host factors are required for equal plasmid segregation during cell division. One of them is cohesin (a multi-subunit protein complex) which mediates sister chromatid cohesion, a crucial mechanism for faithful segregation of replicated chromosomes in eukaryotes. The 2 micron plasmid mimics chromosomes in assembling cohesin at its partitioning locus. Studies on minichromosomes (centromere containing plasmids) reveal that cohesin forms a ring that embraces replicated sister centromeres topologically rather than physically. The functional similarities between chromosome and plasmid segregation prompted us to examine whether the topological mechanism proposed for centromere-mediated replicative cohesion is also true in the case of the plasmid. In the present study, we have characterized the nature and stoichiometry of cohesin's association with the 2 micron plasmid. Another host factor required for equal plasmid segregation is the CenH3 histone variant Cse4, so far considered to be uniquely associated with centromeric nucleosomes. Cse4 provides an epigenetic landmark at centromeres, and is required for assembly of the kinetochore complex. Surprisingly, Cse4 also interacts with the 2 micron plasmid partitioning locus. We have now functionally characterized this interaction, which can be preserved even in an ectopic, chromosomal context. The steady state level of Cse4 is highly limiting in yeast due to ubiquitin-mediated proteolysis. Only centromere-associated Cse4 is protected from this regulatory turnover control. We find that, in contrast to the situation with centromeres, association of Cse4 with the 2 micron plasmid is highly sub-stoichiometric but still promotes equal plasmid segregation. We also find that Cse4 induces an unusual right handed DNA writhe at the plasmid partitioning locus, as it does at the centromere. Our findings suggest that the plasmid has designed strategies to minimize the utilization of host factors that are in short supply. They signify the advantage of clustering and group behavior in the evolutionary success of a multi-copy selfish genome. Finally, they also suggest the possible emergence of the yeast centromere and the plasmid partitioning locus from a common ancestral sequence. / text

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 163-179).

Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 126-147). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

While metabolic enzymes inside the cell nucleus were initially considered “contaminants”, recent evidence has shown that these fulfill essential functions in epigenetic regulation. Indeed a model is emerging in which local metabolite pools influence various nuclear processes. In this model, the subcellular distribution and organization of metabolic factors have a crucial role in the complex logic and regulation of nuclear functions. Cancer cells exploit nuclear metabolic enzymes to alter the synthesis and utilization of metabolites that sustain their transcriptional programs allowing their abnormal proliferation. Understanding the precise molecular mechanisms that modulate the distribution of nuclear metabolic enzymes and their related biological functions has the potential to uncover novel therapeutic vulnerabilities of malignant cells. Here, we describe an unexpected subcellular distribution of acetyl-CoA carboxylase alpha (ACC1), the rate-limiting enzyme of de novo fatty acid synthesis. We found that in cancer cells, ACC1 is not restricted to the cytoplasm. Instead, at mitosis and after the nuclear envelope breakdown, it transiently redistributes into filament-like structures that contact condensed chromosomes. Simultaneous profiling of protein-protein and -DNA interactions defined ACC1 association with different factors associated with the cellular machinery that modulates chromosome segregation, including the centromere, the kinetochore, and the fibrous corona. Inducible depletion of ACC1 resulted in altered mitotic progression and accumulation of chromosome segregation defects – effects that are abolished only with the reconstituted expression of catalytically active mutants of ACC1 but not its inactive counterparts. We further found that the abundance of malonyl-CoA – the main product of ACC1 enzymatic activity – gradually increases towards the onset of mitosis, being a significant determinant for histone malonylation. Overall we uncovered a previously unknown function of ACC1 in modulating mitotic progression and chromosome segregation. Our findings support a model where local niches of malonyl-CoA might act as signal molecules for faithful chromosome segregation.

Faithful chromosome segregation is essential for all living organisms. Bacterial chromosome segregation utilizes highly conserved directional SpoIIIE/FtsK translocases to move large DNA molecules between spatially separated compartments. These translocases employ an accessory DNA-interacting domain (gamma) that dictates the direction of DNA transport by recognizing specific DNA sequences. To date it remains unclear how these translocases use DNA sequence information as a trigger to expend chemical energy (ATP turnover) and thereby power mechanical work (DNA movement). In this thesis, I undertook a mechanistic study of directional DNA movement by SpoIIIE from the Gram-positive model bacterium Bacillus subtilis. Specifically, I was interested in understanding the information transfer within the protein from sequence recognition, to ATP turnover, and ultimately to chromosome translocation. How do DNA sequences trigger directional chromosome movement?

Submitted by Lúcia Bonci Cavalca (l.bonci@hotmail.com) on 2018-04-26T18:08:18Z No. of bitstreams: 1 CAVALCA, LB versão repositorio.pdf: 11866412 bytes, checksum: 0a27e5a7f7f756cb88de16361ad78a27 (MD5) / Approved for entry into archive by Ana Paula Santulo Custódio de Medeiros null (asantulo@rc.unesp.br) on 2018-04-26T19:16:54Z (GMT) No. of bitstreams: 1 cavalca_lb_me_rcla.pdf: 11841916 bytes, checksum: 0d52662f4653c6d3695e6b3ab334549d (MD5) / Made available in DSpace on 2018-04-26T19:16:54Z (GMT). No. of bitstreams: 1 cavalca_lb_me_rcla.pdf: 11841916 bytes, checksum: 0d52662f4653c6d3695e6b3ab334549d (MD5) Previous issue date: 2018-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A cultura de citros é uma das mais importantes do Brasil, sendo a citricultura brasileira a maior do mundo; ainda assim, a produção nacional sofre com pragas e doenças, como cancro cítrico, que afetam sua produtividade. O cancro cítrico é causado pela bactéria Xanthomonas citri subsp. citri (Xac) e tem sido controlado com o uso de estratégias integradas como a eliminação de plantas contaminadas e pulverização de bactericidas cúpricos. O uso de compostos à base de cobre, porém, representa um risco ambiental devido à sua toxicidade e efeito cumulativo, fazendo necessária a investigação de outros compostos com potencial no tratamento fitossanitário contra a doença. Neste estudo avaliamos a atividade bactericida do galato de hexila contra Xac, seu potencial protetivo e curativo no combate ao cancro cítrico, sua fitotoxicidade e predisposição em induzir resistência bacteriana. O composto provocou retardo e diminuição no crescimento populacional de Xac e inibição de seu crescimento in vitro, levando à morte total da população na concentração de 100μg⋅mL-1. A bactéria não foi capaz de desenvolver resistência a Gal-6 ao longo de 31 dias e exibiu taxa de mutantes naturalmente resistentes ao composto menor que 1⋅10-6 para a concentração de 50μg⋅mL-1. A capacidade de germinação de sementes e desenvolvimento de plântulas de rúcula e tomate não foi alterada por Gal-6. A aspersão de galato de hexila em plantas de laranja doce inoculadas com Xac reduziu em até 35% a incidência de sintomas de cancro cítrico, e em até 80% sua severidade. O composto também alterou o comprimento celular de Xac e permeabilidade de membrana. Galato de hexila mostrou-se um bactericida eficaz contra Xanthomonas citri subsp. citri tanto in vitro quanto in planta, além de apresentar baixa fitotoxicidade e baixa probabilidade de indução de resistência em Xac, visto que o composto parece atuar tanto sobre a estrutura física da membrana, quanto sobre o processo de segregação cromossômica/divisão celular bacteriana. / Citrus culture is one of the most important agricultural activities in Brazil, being the country also the biggest producer in the world; nevertheless, this business struggles with pests and diseases, as citrus canker, that affects its profitability. Citrus canker is caused by the bacterium Xanthomonas citri subsp. citri (Xac) and is controlled by using integrated strategies such as elimination of contaminated plants and spraying of cupric bactericides. Using products with copper, however, brings an environmental risk, given 8 its toxicity and cumulative effect, making necessary the research of other compounds with potential to be used as a resource in the phytosanitary treatment against the disease. We evaluated hexyl galate's (Gal-6) bactericidal activity versus Xac and its potential in the preventive and curative treatments against the disease, also Gal-6 phytotoxicity and likeliness to induce bacterial resistance. The compound caused delay and decrease in Xac population growth and in vitro growth inhibition, leading to the total death of the population when at 100 μg⋅mL-1. The bacterium was not able to develop resistance to Gal-6 over 31 days and exhibited a rate of naturally-resistant mutants to the compound of less than 1⋅10-6 at the concentration of 50μg⋅mL-1. Seed germination and seedling development of arugula and tomato were not altered by Gal- 6. Spraying orange plants infected by Xac with hexyl gallate reduced the incidence of citrus canker symptoms by up to 35% and their severity by up to 80%. The compound also altered Xac cell length and membrane permeability. Hexyl gallate proved to be an effective bactericide against Xanthomonas citri subsp. citri both in vitro and in plant, exhibiting low phytotoxicity and low inclination to induce resistance in Xac, given that the compound appears to act on both the physical structure of the membrane and the process of chromosomal segregation/ bacterial cell division.

La mitose assure la répartition égale du génome. La kinase mitotique Aurora B y joue un rôle majeur en contrôlant la fidélité de la ségrégation des chromosomes de par sa localisation aux centromères et aux microtubules, qui nécessite son ubiquitination par CUL3. Cependant, le mécanisme conduisant la forme ubiquitinée d’Aurora B sur ces structures mitotiques reste à déterminer. Dans ce contexte, j’ai pu identifier la protéine UBASH3B, qui contient un domaine de liaison à l’ubiquitine (UBD) comme un régulateur essentiel de la ségrégation chromosomique, agissant comme un récepteur de l’ubiquitine pour Aurora B. UBASH3B interagit directement avec Aurora B et cette interaction est dépendante de la modification d’Aurora B par l’ubiquitine ainsi que de CUL3. UBASH3B ne régule pas le niveau d’expression d’Aurora B. En revanche, UBASH3B se localise aux fuseaux mitotiques et est à la fois nécessaire et suffisant pour transférer Aurora B aux microtubules. De plus, la redistribution d’Aurora B des centromères vers les microtubules contrôle le déroulement et la fidélité de la ségrégation des chromosomes et donc le contenu correct du matériel génétique des cellules. Ainsi, mes résultats expliquent comment la modification par l’ubiquitine régule la localisation et la fonction d’Aurora B, reliant une voie de signalisation impliquant un récepteur à l’ubiquitine à la mitose. / Mitosis ensures equal segregation of the genome. The major mitotic kinase Aurora B controls fidelity of chromosome segregation by its localization to centromeres and microtubules, which requires CUL3-mediated ubiquitylation. However, it remains unknown how ubiquitylated Aurora B is targeted to mitotic structures. Here, I identify ubiquitin-binding domain (UBD) protein UBASH3B that critically regulates chromosome segregation, acting as ubiquitin receptor for Aurora B. UBASH3B directly binds Aurora B, and this interaction is dependent on CUL3 and on ubiquitin recognition. UBASH3B does not regulate protein levels of Aurora B. Instead, UBASH3B localizes to the mitotic spindle and is both required and sufficient to transfer Aurora B to microtubules. Moreover, redistribution of Aurora B from centromeres to microtubules controls timing and fidelity of chromosome segregation and thereby euploidy of cells. Thus, my findings explain how ubiquitin attachment regulates localization and function of Aurora B, linking receptor-mediated ubiquitin signaling to mitosis.

Escherichia coli, comme la majorité des bactéries, possède un unique chromosome circulaire. Au moins une copie du chromosome doit être transmise à chacune des cellules filles avant la division cellulaire afin d'assurer une prolifération cellulaire correcte. Une couplage spatio-temporel précis de la ségrégation avec la division cellulaire est donc nécessaire pour assurer la bonne répartition des deux chromosomes après réplication. La région terminale du chromosome (ter) est la dernière à être répliquée et ségrégée, et migre du pôle vers le centre de la cellule au moment de la mise en place du septum de division, à la fin du cycle cellulaire. Les loci de la région ter présentent une période de cohésion post-réplicative étendue. Cette cohésion étendue est contrôlée par la protéine MatP, qui se fixe spécifiquement au niveau des sites matS, présents uniquement dans ter. MatP se fixe à l'ADN sous forme de dimère, via son domaine N-terminal, et tétramérise via son domaine C-terminal. La tétramérisation est stimulée par la liaison à l'ADN et permet le pontage de deux sites matS distants. MatP interagit aussi avec ZapB, un composant du divisome, la machinerie protéique participant à la formation du septum. Alors que la tétramérisation de MatP semble importante pour la compaction de la région ter, son interaction avec ZapB, qui est localisée au septum via ZapA et FtsZ, participe au positionnement et à la cohésion étendue de cette région. Le couplage de la région ter avec le divisome est essentiel pour le bon déroulement de nombreux évènements tardifs du cycle cellulaire : (i) la ségrégation active, ordonnée et progressive de la région ter par FtsK, un composant du divisome, (ii) la résolution des dimères de chromosomes via la recombinaison spécifique de site XerCD/dif, activée par FtsK, (iii) la résolution des liens d'intercaténation par la TopoIV et (iv) la régulation positive de l'assemblage du divisome en absence des régulateurs négatifs MinCDE et SlmA. Pendant ma thèse, je me suis tout d'abord intéressée au rôle de MatP dans la structuration globale du chromosome. En utilisant un système permettant de visualiser deux loci marqués avec un site parSp1 et un site parSpMT1, reconnu par ParBp1 et ParBpMT1 spécifiquement, nous avons analysé le positionnement et l'orientation du chromosome dans la cellule. Nous avons montré que MatP est nécessaire au positionnement et à l'orientation de tout le chromosome à la fin du cycle cellulaire. La localisation de SlmA dans des souches wt et DeltamatP prouve que l'inactivation de MatP, induisant une mauvais positionnement du chromosome, s'accompagne d'une défaut de localisation de SlmA, et induit donc une inhibition de la division. Ces résultats pris ensemble montre que MatP, SlmA et leur communication à travers la structuration globale du chromosome sont importants pour le management du chromosome et le contrôle de la division cellulaire. En collaboration avec l'équipe d'Olivier Espeli, nous avons utilisé des méthodes de génomiques et de biologie moléculaire pour caractériser la régulation de la TopoIV au cours du cycle cellulaire d'E. coli. Nous avons montré qu'au site dif, les activités de fixation et de clivage de la TopoIV sont améliorées par la présence des recombinases XerCD et de MatP. L'amélioration de l'activité de la TopoIV favorise la décaténation des chromosomes nouvellement répliqués et assure, en lien avec d'autres processus, la séparation précise des chromosomes frères. Ces résultats permettent de mieux comprendre le réseau d'interactions dédiées au management du chromosome à la fin du cycle cellulaire, et l'influence du management du chromosome sur le contrôle de la division cellulaire. / Escherichia coli, as the majority of bacteria, has a unique circular chromosome. Faithfull cell proliferation requires that a least one copy of the chromosome is transmitted to sister cells prior to cell division. A strict temporal and spatial coupling of chromosome segregation with cell division is thus required to ensure the accurate separation of the two fully replicated chromosomes. The terminal region of the chromosome (ter) is the last one to be replicated and segregated, and moves from the pole to the middle of the cell where the division septum is formed, at the end of the cell cycle. Loci of the ter region display an extended cohesion period. This extended cohesion is controlled by the MatP protein, which binds specific matS sites restricted to the ter region. MatP binds DNA as a dimer and forms tetramers via its N-terminal and C-terminal domains respectively. Tetramerisation is stimulated by binding to DNA and pairs remote matS sites. MatP also interacts with ZapB, a component of the divisome, the protein machinery that contributes to septum formation. While tetramerisation of MatP appears important for compacting the ter region, its interaction with ZapB, which is localized at the septum via ZapA and FtsZ, is involved in the positioning and the extended cohesion of this region. The linkage of the ter region with the divisome is required for the success of many later events of the cell cycle : (i) the active, ordered and progressive segregation of the ter region by FtsK, a component of the divisome, (ii) resolution of chromosome dimers via the site-specifique recombination XerCD/dif, activated by FtsK, (iii) the resolution of intercatenation links by TopoIV and (iv) the positive regulation of divisome assembly in the absence of the negative regulators MinCDE and SlmA. During my thesis, I first studied the role of MatP in the chromosome management. By using pairs of loci tagged with parSp1 and parSpMT1 sites recognized by cognate ParB-XFP proteins, we directly analysed chromosome positioning and orientation in the cell. We show that MatP is required for normal positioning and orientation of the whole chromosome at the end of the cell cycle. The localisation of SlmA in wt and Delta matP strains proves that inactivation of MatP leads to inaccuracy of nucleoid positioning accompanied by defects in SlmA localisation, and thus induces division inhibition. Take together, these results show that MatP, SlmA and their interplay are important for chromosome management and control of cell division in E. coli. In collaboration with O. Espeli's team, we have used genomic and molecular biology methods to characterize TopoIV regulation during the E. coli cell cycle. We show that at the dif site, TopoIV binging and cleavage are enhanced by the presence of the XerCD recombinases and MatP. This enhancement of TopoIV activity at dif promotes decatenation of fully replicated chromosomes and ensure, through interaction with other processes, accurate separation of sister chromosomes. These results provide insight into the protein network dedicated to the final step of chromosome management during the cell cycle, and how the chromosome management is linked to cell division.

De nombreuses bananes cultivées et consommées sont des hybrides interspécifiques triploïdes entre Musa acuminata (génome A) et Musa balbisiana (génome B). L'amélioration de ces cultivars nécessite de mettre en place des stratégies complexes liées à leur faible fertilité et leur niveau de ploïdie. De plus, le génome M. balbisiana porteur de caractères agronomiques intéressants est malheureusement porteur de séquences intégrées de Banana streak virus (ou eBSV pour endogenous BSV). Ces eBSV sont capables de produire, dans un contexte de croisements interspécifiques et sous conditions de stress abiotiques, des génomes viraux responsables de l'infection systémique du bananier. L'activation spontanée de ces eBSV est la contrainte majeure des programmes d'amélioration du bananier plantain depuis plus de 10 ans. La ségrégation des chromosomes A et B chez les clones polyploïdes interspécifiques de bananiers est encore très peu connue. Nous avons au cours de cette thèse analysé la recombinaison et la ségrégation chromosomique chez 184 plantes issues de la descendance AAAB (CRBP39) x AA (Pahang_Carbap) au moyen de 38 marqueurs SSR distribués sur les 11 chromosomes Musa et de 6 marqueurs PCR spécifiques des deux espèces BSV présentes chez CRBP39 (eBSGFV-7 et eBSOLV-1). Nous avons observé qu'au cours de la formation des gamètes chez l'allotétraploïde CRBP39, la plupart des marqueurs du tétraploïde AAAB CRBP39 ont une ségrégation de type tétrasomique et que les génomes A et B recombinent au niveau de tous les segments de chromosomes pour lesquels nous pouvions suivre les allèles du chromosome B. D'autre part, nous avons montré que 50% des descendants ont reçu, à un ou quelques loci, un ou trois allèles du parent AAAB (CRBP39) au lieu de deux. La composition allélique de ces gamètes aneuploïdes, la cartographie génétique et l'analyse des corrélations entre marqueurs suggèrent que cette particularité résulte d'une variation structurale entre génomes A et B. Un des chromosomes B correspondrait à une partie des chromosomes 1A et 3A. Nous avons également observé une distorsion de ségrégation des loci eBSV avec une surreprésentation d'individus possédant au moins une intégration eBSV (86%). La régulation des eBSV semble très complexe et nécessitera des études complémentaires pour tenter d'identifier le ou les facteurs génétiques impliqués. Finalement, notre travail a montré que des croisements de type AAAB x AA peuvent générer des plantes possédant du génome B sans aucune intégration BSV (13%). Ce résultat est important car il ouvre une voie de contournement à la contrainte eBSV dans les programmes d'amélioration génétique. / Many cultivated and consumed banana are interspecific triploid hybrids between Musa acuminata (A genome) and Musa balbisiana (B genome). The genetic improvement of these cultivars requires the implementation of complex breeding strategies due to their low fertility and ploidy level. In addition, the B genome of M. balbisiana which bears interesting agronomic traits unfortunately carries endogenous Banana streak virus sequences (eBSV). Under certain conditions such as interspecific crosses and abiotic stresses, these eBSV are able to produce infectious viral genomes responsible for systemic infection of banana. The spontaneous activation of these eBSV is the major constraint of plantains improvement programs for more than 10 years. The A and B chromosomes recombination and segregation in interspecific polyploids banana are still poorly understood. We here analyzed chromosomes recombination and segregation in 184 offspring from the cross AAAB (CRBP39) x AA (Pahang_Carbap) using 38 SSR markers distributed on the 11 Musa chromosomes and 6 specific PCR markers of both BSV species integrated in CRBP39 (eBSGFV-7 and eBSOLV-1). We noticed that during CRBP39 meiosis most of the SSR markers have tetrasomic segregation and that A and B genomes recombine at all chromosomes segments where we were able to follow chromosome B alleles. Besides, we showed that 50% of the offspring received at one or several loci, one or three alleles of the CRBP39 parent instead of two. The allelic composition of these aneuploid gametes, the genetic map and the analysis of correlations between markers suggest that this peculiar observation is due to a structural variation between A and B genomes. One of the B chromosomes would be part of chromosomes 1A and 3A. We also noticed a distorted segregation of eBSV loci with an overrepresentation of individuals harboring at least one of the eBSV (86%). eBSV regulation seems very complex and requires additional studies to identify the genetic factor(s) involved. Finally, we also showed that AAAB x AA crosses can generate plants with B genome but without eBSV. This is the case for 13% of the offspring. This result is important because it shows that we can overcome eBSV constraint in banana breeding programs.