Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 170-189.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 105-116).

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;

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

<p>The ultimate goal of any living cell is to pass on a complete, unaltered copy of its DNA to its daughter cell. The DNA damage response (DDR) and spindle checkpoint are two essential signaling pathways that make it possible for a cell to achieve this goal. The DDR protects genetic integrity by sensing errors in the DNA sequence and activating signaling pathways to arrest the cell cycle and repair the DNA. The spindle checkpoint protects chromosomal integrity by preventing the separation of chromosomes during mitosis until all chromosomes are correctly attached to the mitotic spindle. Proper regulation of both the DDR and the spindle checkpoint is critical for cell survival. In this dissertation I will describe our discovery of novel regulatory mechanisms involved in each of these signaling networks.</p><p>In the first research chapter of this dissertation, we describe our findings concerning how the DDR regulates cyclin F levels. Cyclin F is an F-box protein that associates with the SCF E3 ubiquitin ligase complex to target proteins for degradation. In response to DNA damage, cyclin F levels are downregulated to facilitate increased dNTP production for efficient DNA repair, but the molecular mechanisms regulating this downregulation of cyclin F are largely unknown. We discovered that cyclin F downregulation by the DDR is the combined result of increased protein degradation and decreased mRNA expression. At the level of protein regulation, cyclin F is targeted for proteasomal degradation by the SCF complex. Interestingly, we found that the half-life of cyclin F protein is significantly increased in cells treated with the phosphatase inhibitor calyculin A, which caused cyclin F to be hyper-phosphorylated. Calyculin A also partially prevented cyclin F downregulation following DNA damage. This result suggests that cyclin F phosphorylation stabilizes the protein, and dephosphorylation of cyclin F may be required for its degradation in both unperturbed and DNA damaged cells. We also found that cyclin F downregulation is dependent on the Chk1 kinase, which is predominately activated by the ATR kinase. In examining the mechanism by which Chk1 promotes cyclin F downregulation, we determined that Chk1 represses cyclin F transcription. Lastly, we investigated the role of cyclin F in cell cycle regulation and discovered that both increased and decreased cyclin F expression delay mitotic entry, indicating that an optimal level of cyclin F expression is critical for proper cell cycle progression.</p><p>The second research chapter of this dissertation details our discovery of the requirement for phosphatase activity to inhibit the APC/C E3 ubiquitin ligase during the spindle checkpoint. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C until the chromosomes are properly aligned and attached to the mitotic spindle at metaphase. Once all the chromosomes are properly attached to the spindle, the MCC dissociates, and the APC/C targets cyclin B and securin for degradation so that the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. In our studies, we sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined their effect on the MCC and APC/C activation. Using this approach we found that two phosphatase inhibitors, calyculin A and okadaic acid (1 µM), caused MCC dissociation and APC/C activation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.</p> / Dissertation

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.

La Division mitotique est une phase essentielle du cycle cellulaire qui assure la répartition correcte du contenu génétique. La mitose implique une réorganisation cellulaire profonde qui est principalement induite par une phosphorylation massive de protéines. Cette phosphorylation a lieu grâce à un équilibre fin entre kinases et phosphatases. À l'entrée mitotique, la phosphorylation protéique est induite par l'activation de la kinase cycline B/CDK1 et par l'inhibition de la phosphatase PP2A-B55. Résultats de notre et d'autres laboratoires ont récemment découvert une nouvelle voie essentielle pour moduler la phosphatase PP2A-B55 pendant la transition G2-M. Cette voie inclut la kinase Greatwall (GW) et ses substrats Arpp19 et ENSA. À l'entrée mitotique GW est activé et phosphoryle Arpp19 et ENSA les convertissant en inhibiteurs puissants de PP2A-B55. Étonnamment, aucun autre substrat de GW n'a été identifié jusqu'ici. Cependant, plusieurs éléments suggèrent fortement de nouveaux rôles de GW indépendamment de Arpp19 et de ENSA. L'objectif principal de ce travail était l'identification de nouveaux substrats de GW. À cette fin, j'ai utilisé plusieurs approches, y compris: (1) fractionnement biochimique des lysats de cellules ou des extraits d'oeufs de Xenopus combiné suivi d’une phosphorylation in vitro avec une kinase GW recombinante, (2) SILAC/phosphoproteomique des lysats de cellules exprimant différents niveau de GW, (3) Co-Immunoprecipitation, (4) BioID, et (5) une approche dirigée candidat. Les résultats de la phosphorylation in vitro ont révélé la présence de deux bandes de phosphorylation intéressantes qui sont actuellement analysées. Les deux approches SILAC/phosphoprotéinique et interactome ont révélé l'enrichissement des protéines impliquées dans la régulation post-transcriptionnelle de l'expression génique et des processus liés à l'ARN, une fonction physiologique déjà décrite pour cette voie chez la levure. Enfin, nous avons directement étudié la phosphorylation présumée par GW de trois candidats connus pour être impliqués dans le contrôle du cycle cellulaire. Bien que phosphorylées in vitro par GW, nous n’avons pu identifier le site de phosphorylation que dans l'une de ces trois protéines. Cette protéine, qui correspond à un inhibiteur de phosphatase, semble contrôler la sortie mitotique par la modulation de la déphosphorylation protéique. Un mutant non phosphorylable de cet inhibiteur induit une sortie mitotique perturbée avec une déphosphorylation ralentie des substrats mitotiques et une altération de la dégradation de la cycline B. J’ai pu attribuer ce défaut à une association perturbée de l'inhibiteur avec la phosphatase et, par conséquent, à un timing aberrant de l'inhibition de la phosphatase. Enfin, j'ai identifié le site de phosphorylation par GW comme le facteur clé contrôlant cette association. En résumé, j'ai identifié dans cette étude un nouveau substrat de GW contrôlant l'activité de la phosphatase essentielle pour une division mitotique correcte. / Mitotic division is an essential phase of the cell cycle that ensures the correct repartition of the genetic content. Mitosis involves profound cellular reorganization that is mostly induced by massive protein phosphorylation. This phosphorylation is achieved thanks to the fine-tuning of the balance between kinases and phosphatases. At mitotic entry, protein phosphorylation is induced by the activation of the master kinase Cdk1-cyclin B and the inhibition of the phosphatase PP2A B55. Previous results from our and other laboratories recently discovered a new pathway essential to modulate PP2A-B55 during G2-M transition. This pathway includes the kinase Greatwall (GW) and its substrates Arpp19 and Ensa. At mitotic entry GW is activated and promotes the phosphorylation of Arpp19/Ensa converting them into potent inhibitors of PP2A B55. Surprisingly, no other substrates of GW have been identified so far. However, several pieces of data strongly suggest new roles of GW independently of Arpp19 and Ensa. The main aim of this work was the identification of new substrates of GW. To this end, I used several approaches including: (1) Biochemical fractionation of cell lysates or Xenopus egg extracts combined with in vitro phosphorylation with recombinant GW kinase, (2) SILAC/phosphoproteomics from cell lysates expressing different GW amounts, (3) Co-Immunoprecipitation, (4) BioID and (5) a candidate directed approach. Results from in vitro phosphorylation revealed the presence of two interesting phosphorylated bands that are currently being analysed. Both SILAC/phosphoproteomic and interactome approaches yielded the enrichment of proteins involved post-transcriptional regulation of gene expression and RNA related processes, a physiological function already described for this pathway in yeast. Finally, we directly investigated the putative phosphorylation by GW of three candidates known to be involved in the control of cell cycle. Although phosphorylated in vitro by GW, we could only identify the phosphorylation site in one of these three proteins. This protein, corresponding to a phosphatase inhibitor, appears to control mitotic exit through the modulation of mitotic protein dephosphorylation. A non-phosporylable mutant of this inhibitor promotes a perturbed mitotic exit with delayed dephosphorylation of mitotic substrates and impaired cyclin B degradation. I could attribute this defect to a perturbed association of the inhibitor with the phosphatase and consequently to an aberrant timing of phosphatase inhibition. Finally, I identified the GW phosphorylation site as a key factor controlling this association. In summary, I identified in this study a new substrate of GW controlling phosphatase activity essential for correct mitotic division.

Plusieurs études ont découvert et renforcé l'implication de la dynamique mitochondriale dans le cancer. J'ai découvert un rôle inattendu des protéines kinases de la famille PKD dans la fission mitochondriale. La perte de l'activité PKD a conduit à un blocage de la fission et a entraîné une élongation significative des mitochondries par fusion continue. D'un point de vue mécanique, nous avons montré que les protéines PKD régulent la dynamique mitochondriale en activant le facteur de fission mitochondrial (MFF) par phosphorylation de plusieurs sites. MFF agit comme un récepteur principal de la GTPase DRP1, qui resserre les mitochondries, et il est essentiel à une bonne division mitochondriale. Les trois membres de la famille PKD peuvent phosphoryler MFF. La phosphorylation de MFF est médiée par PKD et la fragmentation mitochondriale se produit pendant la mitose. Comme démontré dans études sur les phosphoprotéomes, la phosphorylation du MFF est augmentée dans les cancers très mitotiques. Ainsi, l'axe de signalisation PKD-MFF régulant la dynamique mitochondriale en mitose pourrait devenir une voie thérapeutique attrayante pour le traitement du cancer. / Over the last two decades, multiple studies have uncovered and strengthen the implication of mitochondrial dynamics in cancer. During my thesis, I discovered an unanticipated role for the PKD kinase family in mitochondrial fission. Loss of PKD activity led to blockade of mitochondrial fission and resulted in a significant elongation of mitochondria by unopposed fusion. Mechanistically, we showed that PKDs regulated mitochondrial dynamics by activating the mitochondrial fission factor (MFF) through phosphorylation of multiple sites. MFF acts as a main receptor for the large GTPase DRP1, which constricts mitochondria, and it is critical for proper mitochondrial division. All three PKD family members could phosphorylate MFF. PKD-mediated MFF phosphorylation and mitochondrial fragmentation occurred specifically during mitosis. As MFF phosphorylation was found to be significantly upregulated in highly mitotic cancers, which was evidenced in several global phosphoproteome studies, the discovered PKD-MFF signaling axis regulating mitochondrial dynamics in mitosis could become an attractive therapeutic avenue for cancer treatment.

Le variant d’histone CENP-A marque épigénétiquement le centromère. La présence de CENP-A au centromère permet le recrutement de protéines centromériques qui constituent la plateforme pour l’assemblage de kinétochores fonctionnels.Dans les cellules humaines, l'extrémité amino-terminale de CENP-A ainsi que la phosphorylation de la sérine 7, ont été signalées comme étant cruciales pour la progression de la mitose. Cependant, aucune phosphorylation de CENP-A dans d'autres espèces de métazoaires n'a été décrite. Ici, nous montrons que le domaine NH2-terminal CENP-A, mais pas sa séquence primaire, est nécessaire pour la mitose dans les fibroblastes embryonnaires de souris (MEFs). Nos données montrent que les défauts mitotiques résultant de la déplétion de CENP-A endogène peuvent être restaurés lorsque les MEFs expriment un mutant GFP-CENP-A dont l'extrémité NH2-terminal de CENP-A a été échangée par la queue phosphorylable de l'histone canonique H3. Inversement, dans ce même mutant, lorsque l’on remplace les deux serines phosphorylables par des résidus alanines, les défauts mitotiques persistent. En outre, le mutant de fusion non- phosphorylable de CENP-A, où les sept serines du domaine NH2-terminal ont été remplacées par des résidus alanines, a été également incapable de restaurer le phénotype mitotique des cellules déplétées en CENP-A endogène.Nous avons également identifié les trois premières sérines de la queue de CENP-A comme sites potentiels de phosphorylation. De plus, nos résultats montrent que l’absence de phosphorylation du domaine amino-terminal conduit à la délocalisation de la protéine centromérique CENP-C. Ces résultats suggèrent que la phosphorylation mitotique de CENP-A est un événement potentiellement fréquent chez les métazoaires et essentiel à la progression mitotique.Dans la seconde partie de ce travail, nous avons voulu lier sans ambiguïté la fonction du domaine NH2-terminal du CENP-A à la mitose. Nous avons conçu une nouvelle méthode, appelée approche Hara-kiri, pour pouvoir éliminer le domaine NH2- terminal seulement pendant la mitose. Ceci afin de répondre à la question ci-dessus dans les cellules humaines. L'élimination du domaine NH2-terminal du CENP-A en utilisant l'approche Hara-kiri en début de mitose a conduit à une augmentation des défauts mitotiques dans les cellules. Prises collectivement, ces données montrent que le domaine NH2-terminal CENP-A est nécessaire pendant la mitose afin d’assurer le bon déroulement de la division cellulaire. / The histone variant CENP-A epigenetically marks the centromere. The presence of CENP-A at the centromeres allows the recruitment of centromeric proteins that constitute the platform for functional kinetochores.In human cells, the NH2-terminus of CENP-A and its phosphorylation at serine 7 in mitosis has been reported to be crucial for the progression of mitosis. However, no phosphorylation of CENP-A in other metazoan species has been described. Here, we show that the NH2-terminus of CENP-A, but not its primary sequence, is required for mitosis in mouse embryonic cells (MEFs). Our data show that the mitotic defects resulting from the depletion of the endogenous CENP-A can be rescued when MEFs expressing a GFP- CENP-A mutant where the NH2-terminus of CENP-A was swapped with the phosphorylatable tail of conventional histone H3. Conversely, no rescue was observed when the two phosphorylatable serines in the H3 tail mutant were replaced with alanines. Furthermore, a non-phosphorylatable fusion mutant of CENP-A where all seven serines in the amino-tail were replaced with alanines, was also unable to rescue the mitotic phenotype of CENP-A depleted cells.We also identified that the first three serines of the tail of CENP-A as potential sites for phosphorylation. Additionally, we were able to link the phosphorylation of CENP-A amino-tail to the proper localization of the key centromeric protein CENP-C. These results suggest that mitotic CENP-A phosphorylation is a potentially common event in metazoans essential for mitotic progression.In the second par of this work we wanted to unambiguously tie the NH2-terminus function of CENP-A to mitosis. To achieve this, we wanted to remove the CENP-A amino-tail only during mitosis and we devised a new method called the Hara-kiri approach in order to answer the above question in human cells. The removal of the NH2-terminal domain of CENP-A using the Hara-kiri approach at the onset of mitosis led to increased mitotic defects in cells. Taken collectively these data show that the CENP-A NH2- terminus is required during mitosis to assure proper cell division.

Cellular checkpoints are typically considered to both facilitate the ordered execution of the cell cycle and to act as a barrier to oncogene driven cell cycles and the transmission of unresolved genetic lesions from one phase to the next. Furthermore, these mechanisms are also believed to underpin the responses of cells, both in normal and cancerous tissues, to those therapies that either directly or indirectly generate DNA damage. In recent studies however, it has become clear these checkpoints permit the passage of significant genomic aberrations into subsequent cell cycle phases and even descendant cells, and that heterogeneous responses are apparent amongst genetically identical cells. The consequences of this checkpoint ‘negligence’ remain relatively uncharacterised despite the importance of checkpoints in current models for how genomic instability is avoided in the face of ubiquitous DNA damage. Unresolved DNA damage is presumably inherited by subsequent cell cycle phases and descendant cells yet characterisation of the consequences of this has been relatively limited to date. I therefore utilised microscopy-based lineage tracing of cells expressing genetically encoded fluorescent sensors, particularly the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Sakaue-Sawano et al., 2008), with semi-automated image analysis to characterise the response of single cells and their descendants to DNA lesions across multiple cell cycle generations. This approach, complemented by generational tracing by flow cytometry, permitted me to characterise the timing of cell fate determination in treated and descendant cells, the non-genetic heterogeneity in checkpoint responses and overall lineage behaviour, correlations between cells (similarly to Sandler et al., 2015) and cell cycle timing dependencies in the response to DNA damaging agents. With these single cell analytical approaches I show that the consequences of DNA damage on descendant cell fate is dramatic, suggesting checkpoint mechanisms may have consequences and even cooperate across phases and generations. U2OS cell lineages traced for three generations following the induction of DNA damage in the form of strand breaks showed greatly induced cell death in the daughters and granddaughters of DNA damaged cells, termed delayed death. Furthermore, lineage behaviour was characterised as highly heterogeneous in when and whether cell death occurred. Complementary flow cytometric approaches validated the findings in U2OS cells and suggested HeLa cells may show similar behaviour. These findings indicate that checkpoint models need to incorporate multigenerational behaviour in order to better describe the response of cells to DNA damage. Understanding the processes governing cell fate determination in descendant cells will impact upon our understanding of the development of genomic instability during carcinogenesis and how DNA-damaging chemotherapeutics drive cells to ‘quit’ the cell cycle.