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Le but de ces travaux de thèse est d’apporter une meilleure compréhension des mécanismes de régulation contrôlant la division cellulaire au niveau moléculaire. La division cellulaire est composée de la mitose et la cytocinèse. Les deux processus doivent être coordonnés étroitement afin de garantir la stabilité du génome. La division cellulaire doit aussi s’équilibrer avec la croissance cellulaire pour que les cellules conservent une taille constante au cours des cycles successifs. La levure S. pombe est un organisme modèle simple très utilisé pour des études de cycle cellulaire et de cytocinèse. Dans ce modèle, nous avons focalisé ce travail de thèse sur les nœuds corticaux médians, des structures protéiques complexes, qui ont une fonction double dans l’engagement en mitose et dans le positionnement du plan de division. Les nœuds médians corticaux sont organisés par la kinase SAD Cdr2. Leur localisation et leur fonction sont régulées négativement pour la DYRK kinase Pom1 qui forme des gradients émanant des extrémités de la cellule. Les nœuds corticaux médians contiennent une voie d’inhibition pour Wee1 qui promeut l’entrée en mitose. Cette voie implique la kinase SAD Cdr1, un inhibiteur direct de Wee1 et pourrait coupler l’entrée en mitose à la taille de la cellule par levée progressive de l’inhibition exercée par Pom1 quand les cellules s’allongent. Cdr2 recrute aussi l’anillin Mid1 sur les nœuds corticaux médians ainsi qu’une série de composants additionnels, Blt1, Gef2, Nod1 et Klp8, pour former des précurseurs médians de l’anneau contractile de cytocinèse qui se compactent en un anneau fin pendant la mitose. La localisation médiane des nœuds, contrôlée négativement par les gradients polaires de Pom1 prédéfinit ainsi le plan de division au centre géométrique de la cellule. Dans la première partie de ma thèse, j’ai étudié la protéine des nœuds corticaux médians Blt1 dont la fonction restait énigmatique. Nous avons montré que Blt1 promeut une association robuste de Mid1 avec les nœuds corticaux. Blt1 interagit avec Mid1 via le RhoGEF Gef2 pour stabiliser les nœuds au cortex cellulaire durant les premiers stades de l’assemblage de l’anneau contractile. L’extrémité N-terminale de Blt1 est nécessaire à sa localisation ainsi qu’à sa fonction, tandis que son extrémité C-terminale favorise sa localisation au cortex en interagissant avec des phospholipides. Dans des cellules dans lesquelles ni Mid1 ni Blt1 ne peuvent s’attacher à la membrane, les nœuds se détachent du cortex et génèrent des anneaux contractiles de cytocinèse aberrants. Nous en avons conclu que Blt1 agit comme une protéine d’échafaudage pour les précurseurs de l’anneau contractile, et que Blt1 et Mid1 constituent des ancres membranaires redondantes pour le positionnement du plan de division. Dans une deuxième partie de ma thèse, j’ai étudié comment Cdr2 organise les différents composants des nœuds en voies fonctionnelles qui favorisent l’entrée en mitose et la division médiane. J’ai montré que l’interaction de Cdr2 avec Wee1 et Mid1 dépend du domaine UBA de Cdr2 de manière dépendante de l’activité kinase. En revanche, Cdr1 s’associe avec l’extrémité C-terminale de Cdr2, composée des domaines basique et KA1 d’association aux lipides membranaires. De manière intéressante, Mid1 interagit également avec l’extrémité C-terminale de Cdr2 et pourrait ponter les parties N- et C-terminales de Cdr2, alors que Blt1 s’associe à la région centrale de Cdr2. Nous faisons l’hypothèse que l’association des effecteurs de Cdr2 avec différents domaines de Cdr2 pourraient contraindre Cdr1 et Wee1 spatialement pour promouvoir l'inhibition de Wee1 quand la kinase Cdr2 est active. / The aim of this PhD work is to bring a better understanding of the regulatory mechanism controlling cell division in space and time at the molecular level. Cell division is composed of mitosis and cytokinesis. Both processes need to be perfectly coordinated in order to guarantee genome integrity. Cell division also needs to be properly balanced with cell growth to maintain cell size constant during successive cell cycles. Temporal and spatial regulatory mechanisms ensure the coordination of these events. The fission yeast Schizosaccharomyces pombe is a simple rod-shaped model organism well-known for cell cycle and cytokinesis studies. In this model, we focused the work of this thesis on the medial cortical nodes, complexe protein structures that have a dual role in mitotic commitment and in division plane positioning. Medial cortical nodes are organized by the SAD kinase Cdr2. Their localization and function is negatively regulated by the DYRK kinase Pom1 that forms a gradient emanating from the cell tips. Medial cortical nodes contain an inhibitory pathway for Wee1, promoting mitotic entry. This pathway involves the SAD kinase Cdr1, a direct inhibitor of Wee1 and has been proposed to couple mitotic entry to cell size by progressive alleviation of Pom1 inhibition when cells grow longer. Cdr2 also recruits to medial nodes the anillin Mid1 as well as a series of four additional components, Blt1, Gef2, Nod1 and Klp8, to form medial precursors for the cytokinetic contractile ring that compact into a tight ring during mitosis. Nodes medial localization, negatively controlled by Pom1 gradients, predefines thereby the division plane in the cell geometrical center. In a first part of my thesis, I studied the previously enigmatic cortical node protein Blt1. We showed that Blt1 promotes the robust association of Mid1 with cortical nodes. Blt1 interacts with Mid1 through the RhoGEF Gef2 to stabilize nodes at the cell cortex during the early stages of contractile ring assembly. The Blt1 N terminus is required for localization and function, while the Blt1 C terminus promotes cortical localization by interacting with phospholipids. In cells lacking membrane binding by both Mid1 and Blt1, nodes detach from the cell cortex and generate aberrant cytokinetic rings. We conclude that Blt1 acts as a scaffolding protein for precursors of the cytokinetic ring and that Blt1 and Mid1 provide overlapping membrane anchors for proper division plane positioning. In the second part of my thesis, I studied how Cdr2 scaffolds various nodes components to organize them in functional pathways promoting mitotic commitment and medial division. I showed that Cdr2 interaction with Wee1 and Mid1, depends on Cdr2 UBA domain in a kinase activity dependent manner. In contrast, Cdr1 associates with Cdr2 C-terminus composed of basic and KA-1 lipid-binding domains. Interestingly, Mid1 also interacts with Cdr2 C-terminus and may the bridge N- and C-terminal domains of Cdr2 while Blt1 associates with the central spacer region. We propose that the association of Cdr2 effectors with different Cdr2 domains may constrain Cdr1 and Wee1 spatially to promote Wee1 inhibition upon Cdr2 kinase activation.

La vida aeróbica conlleva la formación de especies reactivas derivadas del oxígeno: el radical hidroxilo (OH·), el ión superóxido (O2·-) y el peróxido de hidrógeno (H2O2). En Schizosaccharomyces pombe, dos rutas controlan las respuestas antioxidantes en respuesta a estrés oxidativo por H2O2: la del factor de transcripción Pap1 y la de la MAP quinasa Sty1. En esta tesis doctoral, hemos determinado que la activación de Pap1 se da en respuesta a dosis moderadas, pero no severas, de H2O2. Hemos identificado a la peroxirredoxina Tpx1 como sensor y transmisor de la señal de estrés oxidativo a Pap1. La inactivación temporal de Tpx1, durante estrés oxidativo severo, por oxidación a sulfínico de su cisteína catalítica inhibe la transmisión de señal a Pap1. En dichas condiciones, se activa la ruta de Sty1, que media la inducción de Srx1, cuya función es reducir y, con ello, reactivar a Tpx1. Finalmente, hemos estudiado el papel esencial de Tpx1 en aerobiosis. / Aerobic life involves formation of reactive oxygen species: hydroxyl radical (OH·), superoxide ion (O2·-) and hydrogen peroxide (H2O2). In Schizosaccharomyces pombe, two pathways respond to H2O2 and trigger independent antioxidant-gene responses: the Pap1 and the Sty1 pathways. In this thesis project, we have determined that the activation of the transcription factor Pap1 occurs only at low, but not elevated, H2O2 concentrations. We have identified the peroxiredoxin Tpx1 as a H2O2-sensor and redox activator of Pap1. The temporal inactivation of Tpx1 during severe oxidative stress, by oxidation of its catalytic cysteine to sulfinic acid, inhibits signal transduction to Pap1. During these conditions, the MAP kinase Sty1 is activated and expression of the sulfiredoxin Srx1 is triggered. Srx1 functions to reduce and thus reactivate Tpx1. Finally, we have analysed the essential function of Tpx1 in aerobiosis.

The meiotic cell cycle is modified from the mitotic cell cycle by having a premeiotic S phase which leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis, and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is expressed only during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show a decreased intragenic meiotic recombination and a delay at the onset of meiosis I. When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding for two proteins with different function depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimeras show a meiotic-specific regulation of splicing, exactly as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 and Fkh2. While Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and pre-meiotic S phase. / El ciclo meiótico se diferencia del ciclo mitótico por tener una fase S pre-meiótica caracterizada por altos niveles de recombinación, dos rondas de división nuclear sin síntesis de DNA entre las dos y una segregación cromosómica reduccional. Rem1 es una ciclina que sólo se expresa en meiosis en la levadura de fisión Schizosaccharomyces pombe. Celulas con rem1 deleccionado presentan una tasa de recombinación intragénica disminuida y un retraso en el inicio de meiosis I. Cuando se expresa ectópicamente en células creciendo vegetativamente, Rem1 induce un arresto en G1 seguido de catástrofe mitótica. Este trabajo describe que la expresión de rem1 está regulada a nivel de la trascripción y el procesamiento, codificando para dos proteínas con funciones diferentes dependiendo de la retención intrónica.. Hemos determinado que la regulación del splicing de rem1 no depende de ninguna región transcrita del gen. Además, cuando el promotor se fusiona a otros genes que contienen intrones, las quimeras presentan una regulación específica de meiosis como el rem1 endógeno. Esta regulación depende de dos factores de transcripción de la familia Forkhead, Mei4 y Fkh2. Mientras Mei4 induce la transcripción y el splicing de rem1, Fkh2 es responsable de la retención intrónica del tránscrito durante crecimiento vegetativo y fase S pre-meiótica.

Notch pathway plays a critical role during the development and life of metazoan organisms. CSL proteins are the component of the Notch pathway that mediates the regulation of target genes. The discovery of CSL-like proteins in yeast raised the question of their function in unicellular organisms which did not utilize the canonical Notch pathway. CSL-homologues in yeast are conserved in parts that are important for DNA binding and for fission yeast proteins it was shown that they bind to CSL recognition elements in vitro. In fission yeast, CSL paralogues Cbf11 and Cbf12 play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Yeast CSL proteins have long and intrinsically unstructured N- terminal domains compared to metazoan CSL proteins. In this study, we investigated the functional significance of these extended N-termini of CSL proteins by their complete removal. For newly constructed truncated variants of proteins Cbf11 and Cbf12 in Schizosaccharomyces pombe we observed the lack of ability to bind CSL recognition RBP probe. The removal of N-terminal parts of CSL proteins in fission yeast led to the change in their cellular localization. Once strongly preferred nuclear localization changed by the removal of N-terminal domains to cytoplasmic localization with a...

Kinase Src plays an essential role in signal transduction from activated surface receptors. Src is involved in signal pathways that participate in the control of cell proliferation, differentiation or motility. That is why Src activation undergoes strict and complex regulation. Inactive conformation is maintained by intramolecular inhibitory interactions. SH3 domain associates with a polyprolin helix in CD linker whereas SH2 domain binds phosphorylated C-terminal tyrosine 527. Both regulatory domains maintain contacts with the lobes of a kinase domain thereby stabilizing an inactive conformation of the catalytic domain. Transition to an active state is accompanied by a disruption of these inhibitory interactions. Conformation changes are substantially influenced by the phosphorylation status of key tyrosines 416 and 527. Phosphoproteomic analysis revealed new Src tyrosine residue, which can be phosphorylated in vivo. It has been found, that tyrosine works as an additional regulator of Src activity. This is Tyr 90, which forms one of the hydrophobic pockets in the binding surface of Src SH3 domain. Based on the expression of phosphomimic mutant Src 90E in S. pombe or in SYF lineage, it has been observed, that Tyr 90 phosphorylation elevates Src kinase activity. The reason is that the phosphate...

Thesis advisor: Charles S. Hoffman / Glucose detection in yeast occurs via a cAMP signaling pathway that is similar to that of other signaling pathways in humans. The presence of glucose in the environment ultimately represses, as a result of cAMP signaling, the transcription of the gene fbp1. Adenylate cyclase is known to convert ATP to cAMP, and is thus a central protein in the propagation of the signal. Mutant forms of the adenylate cyclase gene (git2) have been found by the inability for the organism to repress fbp1 transcription in the presence of glucose. In this study, two questions were under investigation. The first was focused on the ability of the mutations to affect the dimerization of the catalytic domain. The second investigated multiple protein-protein interactions in the leucine rich-repeat (LRR) domain of adenylate cyclase. Both domains contain mutations that confer an activation defect, and they are thus are thought to have a relationship. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

In order to generate healthy daughter cells, nuclear division and cytokinesis need to be coordinated. Premature division of the cytoplasm in the absence of chromosome segregation or nuclear proliferation without cytokinesis might lead to aneuploidy and cancer. The cyclin dependent kinases, CDKs, are a main regulator of the cell cycle. Timely increase and decrease in their activity is required for cell cycle progression. To enter mitosis, mitotic CDK activity needs to rise. CDK activity stays elevated until chromosome segregation is completed and exit from mitosis requires decrease in CDK activity. Observations in several experimental systems suggest that coordination of cytokinesis with the nuclear cycle is regulated through CDK activity. Prolonged high CDK activity, as it occurs when chromosome segregation is delayed, was found to oppose cytokinesis. Prevention of cytokinesis through high CDK activity may therefore provide a mechanism to prevent precocious cell division in the absence of chromosome segregation. To prevent polyploidy when cell division is delayed, progression through the next nuclear cycle should be inhibited until cytokinesis is completed, presumably by the inhibition of CDK activity. In the fission yeast Schizosaccharomyces pombe, a signaling cascade called Septation Initiation Network (SIN) is required for the coordination of cytokinesis with the nuclear cycle. The SIN is essential for cytokinesis, triggering the execution of cell division through constriction of the actomyosin ring. The activation of the SIN signaling cascade, and thus cytokinesis, is opposed by high CDK activity, preventing precocious cytokinesis. S. pombe delay entry into the next nuclear division in response to delayed cytokinesis due to defects in the contractile ring until cytokinesis is completed thereby preventing the accumulation of multinucleate, non viable cells. This safeguard against multinucleate cells is termed the cytokinesis checkpoint. The cytokinesis checkpoint keeps CDK activity low, preventing nuclear cycle progression. The SIN is required for the cytokinesis checkpoint and therefore is a key coordinator between nuclear cycle and cytokinesis. How the SIN functions in the cytokinesis checkpoint was not known. Cdc14-family phosphatases are highly conserved from yeast to humans, but were only characterized in Saccharomyces cerevisiae at the time this thesis was initiated. Cdc14 had been identified as the effector of a signaling cascade homologous to the SIN, called the mitotic exit network (MEN), which is required for exit from mitosis. This thesis describes the identification of the S. pombe Cdc14-like phosphatase Clp1p as a component of the cytokinesis checkpoint. Clp1p opposes CDK activity, and Clp1p and the SIN activate each other in a positive feedback loop. This maintains an active cytokinesis checkpoint and delays mitotic entry. We further found that Clp1p regulates chromosome segregation. Concluding, this thesis describes discoveries adding to the characterization of the cytokinesis checkpoint and the function of Clp1p. While others found that Cdc14-family phosphatases, including Clp1p, have similar catalytic functions, we show that their biological function may be quite different between organisms, possibly due to different biological challenges.

Establishment and maintenacne of a polarized axis is essential for all organisms. Cells can either change their shape in response to extracellular cues or maintain a stable polarity axis via landmarks defined in relation to internal cues. In the fission yeast Schizosaccharomyces pombe,microtubules regulate cortical cell polarity together with the landmark protein Tea1. Tea1 is transported to cell tips on microtubule plus-­‐ends and deposited upon microtubule contact with the membrane. Although Tea1 has been shown to interact with several binding-­partners, Tea1 anchoring at the cell tip depends mostly on the membrane-­associated protein, Mod5. Tea1 and Mod5 accumulate in clusters at the cell tip in a mutually dependent manner. I used a combination of live-­‐cell imaging, FRAP (Fluorescence Recovery After Photobleaching) and computational modeling to dissect the dynamics of the Tea1-­‐Mod5 interaction. I have shown that although Tea1 is stably associated with the cell tip, Mod5 is mobile within the cell tip. I proposed a model in which Tea1 is stable at the cell tip due to self-­‐polymerization and association in the form of a cluster-­‐network. In the model, the role of Mod5 in the cluster-­‐network is to facilitate the formation of Tea1-­‐Tea1 interactions. Moreover, in the model, Mod5 is restricted to the cell tip due to iterative binding to and release from the Tea1 cluster-­‐network. The properties of the proposed Tea1 cluster-­‐ network might contribute to the behavior of Tea1 as a polarity landmark. I hypothesized that Tea1 transfer from the microtubules to the cell tip was regulated by phosphorylation. Tea1 phosphorylated residues were mapped using mass spectroscopy (MS), and identified to be mostly enriched within a central region of the protein. Using a combination of mutagenic analysis and live-­‐cell imaging I demonstrate that Tea1 phosphorylation might be required for its dissociation from the cluster-­‐network at the cell tip. This suggests that Tea1 interactions within the cluster network are phospho-­‐regulated by one of the several tip-­‐localized kinases. It has been shown in other organisms and in this thesis that comparison among MS samples requires quantitative MS methodologies. Thus, I developed a robust SILAC (Stable Isotope Labeling in Cell Culture) method to perform quantitative MS in S. pombe. As a proof-­‐of-­‐principle of the method I performed a proteome-­‐wide comparison between the late G2 and the G1/S transition of the cell cycle. The cell cycle proteome-­‐wide analysis not only quantified variation in expression levels of cell cycle regulated proteins but also identified novel cell cycle regulated proteins. It has been previously shown that Tea1, Tea3 and Mod5 can interact simultaneously, with each pair interacting independently of the third protein. I describe here a Mod5 mutagenic analysis screen designed to separate Tea1 and Tea3 binding site on Mod5. The Mod5-­‐mutants obtained from this analysis indicate that the Tea3-­‐Mod5 interaction may play a role in cell polarity establishment. Moreover, although Tea3 is non-­‐essential for the cluster-­‐network formation, Tea3 might be important for its compaction, which may be particularly important during de novo formation of cell polarity.