The Golgi apparatus is organised as a 'ribbon' of interconnected stacks of membranous cisternae that have characteristic enzyme compositions that define where cargo proteins are glycosylated during their transport towards the plasma membrane in mammalian cells. There are two classes of Golgi transport schemes: one based on stable cisternae, and the other on 'maturing' cisternae. With the stable cisternae model, the Golgi resident proteins (e.g., glycosylating enzymes) always remain in the same cisterna and the secretory cargo is transported forwards in vesicles, to move progressively from one cisterna to the next. According to the maturation scheme, new cisternae form at the eis-Golgi face, and then 'move forward' through the stack; at the same time, the cisternae 'mature' by acquiring, and then losing, the glycosylating enzymes, which recycle backwards in step with cargo progression. Here, the cargo remains stable within the cisterna, and is transported forwards within the advancing cisternae. While the maturation mechanism has significant experimental support, the crucial evidence that Golgi enzymes recycle through the stack in mammalian cells remains missing. In the present study, I designed experiments based on the use of Golgi-resident enzyme constructs that can be reversibly polymerised to distinguish the maturing versus stable cisternae models. The data strongly fit with the predictions of the cisternal maturation model

Cell division in almost all bacteria is orchestrated by the essential tubulin homologue FtsZ, which assembles into a ring-like structure and acts as a scaffold for the division machinery. Bacterial cell division was recently validated as an important target for new antibiotics by the demonstration that low molecular weight inhibitors of FtsZ, called benzamides, can cure mice infected with a lethal dose of Staphylococcus aureus. In order to understand the mode of action of the benzamides, a detailed cytological and biochemical analysis of their effects on cell division has been performed in the Gram-positive model organisms, Bacillus subtilis and S. aureus. In treated cells of B. subtilis FtsZ assembles into foci throughout the cell, including abnormal locations at the cell poles and over the nucleoid. These foci are not inactive aggregates as they remain dynamic, turning over almost as rapidly as untreated polymers. Remarkably, although division is completely blocked, the foci efficiently recruit division proteins that normally eo-assemble with FtsZ. However, they show no affinity for components of the Min or Nucleoid Occlusion systems, which normally act to spatially restrict FtsZ assembly. In vitro, the benzamides strongly promote the polymerisation of FtsZ, into hyper-stable polymers, which are highly curved. Importantly, even at low concentrations, benzamides transform the structure of the Z ring, resulting in abnormal helical cell division events. Furthermore, the characterisation of compound resistant mutants, and the isolation of a benzamide-dependent strain of B. subtilis, has revealed novel insights into the nature of Z ring assembly and the likely mechanisms by which the benzamides may act. Finally, the Nucleoid Occlusion factor Noc, which is a potent inhibitor of cell division, has been characterised in vitro and its ability to interact with, and modulate the assembly of FtsZ, examined.

Cell cycle events are the basis of cell duplication and division. Control of the cell cycle involves various mechanisms, which appear conserved throughout eukaryotic evolution. Among them, transcription plays a major part. Studies on gene expression in two yeast model organisms, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Identified waves of transcription through the cell cycle, which require specific transcription factor complexes binding to promoter sequences. These periodic expression patterns are clustered corresponding to the four main cell cycle transitions, suggesting that their serial regulation is interdependent. Cytokinesis occurs at the end of the cell cycle, and includes formation of the actomyosin ring, synthesis of a membrane barrier and the eventual splitting of mother- daughter cells. In S. pombe, genes that are transcriptionally regulated during the M/G1 transition encode many of the components required for cytokinesis. Previously, we identified a transcription factor complex, which we named the PBF, and a promoter motif, the PCB, that together control M/G1 gene expression in S. pombe. In this thesis, we provide evidence for three transcription factors, Fkh2p, Sep1p and Mbx1p, as components of PBF and describe the isolation a Fkh2p-interacting protein, which we named Lyn1p. We also show that Fkh2p and Mbx1p are both hosphoproteins and present data that a polo-like kinase, Plo1p, phosphorylates Mbx1p In vitro. Furthermore, we show that Plo1p controls M/G1 gene expression, and is itself regulated by the PBF-PCB complex. Recent reports identified a glycolytic enzyme, Tdh1p, as a component of the Mediator complex, which bridges the transcription activating proteins to the RNA polymerase II (RNAP II) machinery that controls RNA production. Here, we present evidence that Sep1p interacts with Tdh1p, therefore suggesting a mechanism by which PBF regulates M/G1- specific gene expression, through direct interaction with the RNAP II machinery. In summary, we have characterised the molecular mechanism that controls M/G1 transcription in S. pombe. This involves Plo1p phosphorylating and controlling the PBF transcription factor complex, which in turn interacts with RNAP II machinery to control specific gene expression, and eventually cytokinesis and cell separation.

L’implication et les rôles de la PKA et de la MAPK/ERK lors de la division cellulaire, ont fait l’objet de nombreuses études. Pourtant les profils spatio-temporels des activités de ces kinases au cours des différentes étapes du cycle et notamment lors de la mitose sont controversés et restent à éclaircir. Le but de ce travail a été la détermination de ces profils grâce à l’utilisation et au développement d’outils moléculaires basés sur des propriétés de la fluorescence, capables de rapporter l’activité kinase in vivo, qui sont appelés biosenseurs FRET. Nous avons mis en évidence que l’activité de PKA augmente lors de la mitose pour ensuite chuter rapidement lors de la cytokinèse dans les cellules HeLa. Lors de la métaphase et de l’anaphase, l'activité de PKA est particulièrement élevée à proximité des chromosomes et ce, indépendamment d’une relocalisation de ses sous-unités catalytiques. De plus, l’utilisation d’inhibiteur de PKA conduit à l’apparition de phénotypes mitotiques aberrants, indiquant le rôle essentiel de cette augmentation d’activité dans le maintien de l’intégrité du génome. Ces phénotypes sont similaires à ceux décrits pour des perturbations de l’activité de MAPK/ERK. Le développement d’un biosenseur FRET optimisé pour les mesures d’activité de MAPK/ERK nous a permis de déterminer que son activité globale ne varie pas lors de la mitose mais connait en revanche une diminution forte et très brève lors de la cytokinèse. L’inhibition de PKA induit une augmentation sensible de la phosphorylation de MAPK/ERK, ce qui pourrait suggérer ainsi un lien entre les activités de ces deux protéines dans la répartition correcte du matériel génétique lors de la mitose. / Even if the roles and contribution of PKA and MAPK/ERK in cell cycle have been the topic of several studies, the spatio-temporal profiles of their activities are still controversial and remain to be clarified. The aim of my PhD was to highlight those activity profiles during the cell cycle in HeLa cells, by using or developing new molecular tools, based on fluorescence properties that are able to report kinase activity in vivo and named FRET-based biosensors.The use of these biosensors allowed us to reveal that PKA activity increased at the onset of mitosis and stayed high until the completion of cytokinesis. During metaphase and anaphase, this activity was especially high in the close vicinity of the condensed chromosomes, independently of any concomitant relocalization of PKA catalytic sub-units within the cell. Moreover inhibition of PKA activity during mitosis lead to improper mitotic phenotype (i.e. : misalignment of the DNA on the spindle, precocious segregation of part of the chromosomes), pointing out the essential role of the activity increase in genetic stability. Those observed phenotypes are similar to those described upon experimental modifications of the MAPK/Erk activity level. By means of the development of a new improved MAPK/Erk activity biosensor, we showed that its global activity does not change during mitosis, but goes through a brief and strong decrease during cytokinesis. As the inhibition of PKA induces a noticeable increase of MAPK/Erk phosphorylation, those results could suggest a link between those two kinases activities in the correct distribution of the DNA to daughter cells during mitosis.

Entry into mitosis in eukaryotes depends on the activation of the Cyclin-dependent kinase 1 (Cdk1), which phosphorylates many mitotic protein substrates. Activation of Cdk1 requires formation of a complex with Cyclin B (CycB), which gradually rises in concentration during interphase. However, in most organisms Cdk1 activation is not gradual but switch-like, because phosphorylation of the Cdk1-CycB complex by the Wee1 kinase normally keeps Cdk1-CycB inactive during interphase. Mitotic entry is induced when rapid dephosphorylation of Cdk1-CycB by the Cdc25 phosphatase causes abrupt activation of Cdk1-CycB. Cdk1-CycB in turn phosphorylates both Wee1 and Cdc25 leading to Cdc25 activation and Wee1 inhibition. This regulation creates both a positive and a double-negative feedback loop in the system, which are thought to generate a sharp, bistable switch that controls mitotic entry. Bistability is known to require positive feedback and ultrasensitivity, however, how ultrasensitivity arises in the mitotic switch is subject to extensive research efforts both experimentally and theoretically. In this thesis I explore several possible sources of ultrasensitivity in the mitotic switch through mathematical modelling. Based on theoretical considerations and experimental evidence, I show that the existence of multiple positive feedback loops, multisite phosphorylation, and Cdk1-CycB-dependent regulation of Cdk1-counteracting phosphatase activity can all contribute to ultrasensitivity and bistability in the mitotic switch. I analyse models of the mitotic switch including these bistability-generating mechanisms, to simulate and explain experimental data and make testable predictions. I argue that it is unlikely that a single mechanism is responsible for ultrasensitivity in this system, and that bistability requires a combination of different sources, including the ones studied here and others such as enzyme saturation and sequestration effects. I also highlight the importance of network architecture and coherent regulation of opposing reactions in generating efficient biochemical switches. Finally, I draw on recent experimental evidence and ideas derived from this analysis to propose a revised network of the mitotic switch.

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