Most existing studies of receptor signaling are qualitative, which can lead scientists to misinterpret or overlook key information about the extent and timing of key events. To overcome these shortcomings, we have applied quantitative approaches to characterize receptor activation and signaling events. Most signaling studies focus on events occurring at a particular level in the system (e.g., on the membrane, at the level of phosphorylation of intracellular signaling molecules, or at the level of transcription). Instead, we are interested in taking a longitudinal view of signaling by achieving a quantitative understanding of a single signaling pathway from initial stimulation of the receptor by its growth factor (GF) ligand, through to gene expression, and functional cellular responses. As a model system for our studies, we used the growth factor receptor tyrosine kinase, REarranged during Transfection (RET), which requires a ligand and a glycosylphosphatidylinositol-anchored co-receptor for activation. RET mediates the response of cells to members of the glial cell-line derived neurotrophic factor (GDNF) family of neurotrophins, which are important in the development and maintenance of a subset of neuronal cells as well as in other cell types and tissues. We have characterized the molecular mechanisms of RET activation and signaling by pursuing the following four aims: 1) We developed a sensitive and robust luciferase reporter gene assay for RET signaling. 2) We characterized the dynamic relationship between receptor activation and downstream signaling events, including gene transcription and translation of three target genes. 3) We used the reporter gene assay, and other detection approaches, to test and quantify crosstalk between RET and other GF receptors. 4) We developed a FRET reporter system to enable monitoring of the assembly of the activated RET receptor complex on cells, as a means to distinguish between ligand-induced oligomerization and pre-associated oligomer mechanisms. Through these four aims, we have established new methods to quantitatively elucidate mechanisms of GF receptor activation, new insights into how signals are propagated from the receptor to the nucleus and into a functional response, and have established crosstalk between RET and other GF receptor pathways.

Les changements évolutifs nous instruisent sur les nombreuses innovations permettant à chaque organisme de maximiser ses aptitudes en choisissant le partenaire approprié, telles que les caractéristiques sexuelles secondaires, les patrons comportementaux, les attractifs chimiques et les mécanismes sensoriels y répondant. L'haploïde de la levure Saccharomyces cerevisiae distingue son partenaire en interprétant le gradient de la concentration d'une phéromone sécrétée par les partenaires potentiels grâce à un réseau de protéines signalétiques de type kinase activées par la mitose (MAPK). La décision de la liaison sexuelle chez la levure est un événement en "tout–ourien", à la manière d'un interrupteur. Les cellules haploïdes choisissent leur partenaire sexuel en fonction de la concentration de phéromones qu’il produit. Seul le partenaire à proximité sécrétant des concentrations de phéromones égales ou supérieures à une concentration critique est retenu. Les faibles signaux de phéromones sont attribués à des partenaires pouvant mener à des accouplements infructueux. Notre compréhension du mécanisme moléculaire contrôlant cet interrupteur de la décision d'accouplement reste encore mince. Dans le cadre de la présente thèse, je démontre que le mécanisme de décision de la liaison sexuelle provient de la compétition pour le contrôle de l'état de phosphorylation de quatre sites sur la protéine d'échafaudage Ste5, entre la MAPK, Fus3, et la phosphatase,Ptc1. Cette compétition résulte en la dissociation de type « intérupteur » entre Fus3 et Ste5, nécessaire à la prise de décision d'accouplement en "tout-ou-rien". Ainsi, la décision de la liaison sexuelle s'effectue à une étape précoce de la voie de réponse aux phéromones et se produit rapidement, peut-être dans le but de prévenir la perte d’un partenaire potentiel. Nous argumentons que l'architecture du circuit Fus3-Ste5-Ptc1 génère un mécanisme inédit d'ultrasensibilité, ressemblant à "l'ultrasensibilité d'ordre zéro", qui résiste aux variations de concentration de ces protéines. Cette robustesse assure que l'accouplement puisse se produire en dépit de la stochasticité cellulaire ou de variations génétiques entre individus.Je démontre, par la suite, qu'un évènement précoce en réponse aux signaux extracellulaires recrutant Ste5 à la membrane plasmique est également ultrasensible à l'augmentation de la concentration de phéromones et que cette ultrasensibilité est engendrée par la déphosphorylation de huit phosphosites en N-terminal sur Ste5 par la phosphatase Ptc1 lorsqu'elle est associée à Ste5 via la protéine polarisante, Bem1. L'interférence dans ce mécanisme provoque une perte de l'ultrasensibilité et réduit, du même coup, l'amplitude et la fidélité de la voie de réponse aux phéromones à la stimulation. Ces changements se reflètent en une réduction de la fidélité et de la précision de la morphologie attribuable à la réponse d'accouplement. La polarisation dans l'assemblage du complexe protéique à la surface de la membrane plasmique est un thème général persistant dans tous les organismes, de la bactérie à l'humain. Un tel complexe est en mesure d'accroître l'efficacité, la fidélité et la spécificité de la transmission du signal. L'ensemble de nos découvertes démontre que l'ultrasensibilité, la précision et la robustesse de la réponse aux phéromones découlent de la régulation de la phosphorylation stoichiométrique de deux groupes de phosphosites sur Ste5, par la phosphatase Ptc1, un groupe effectuant le recrutement ultrasensible de Ste5 à la membrane et un autre incitant la dissociation et l'activation ultrasensible de la MAPK terminal Fus3. Le rôle modulateur de Ste5 dans la décision de la destinée cellulaire étend le répertoire fonctionnel des protéines d'échafaudage bien au-delà de l'accessoire dans la spécificité et l'efficacité des traitements de l'information. La régulation de la dynamique des caractères signal-réponse à travers une telle régulation modulaire des groupes de phosphosites sur des protéines d'échafaudage combinées à l'assemblage à la membrane peut être un moyen général par lequel la polarisation du destin cellulaire est obtenue. Des mécanismes similaires peuvent contrôler les décisions cellulaires dans les organismes complexes et peuvent être compromis dans des dérèglements cellulaires, tel que le cancer. Finalement, sur un thème relié, je présente la découverte d'un nouveau mécanisme où le seuil de la concentration de phéromones est contrôlé par une voie sensorielle de nutriments, ajustant, de cette manière, le point prédéterminé dans lequel la quantité et la qualité des nutriments accessibles dans l'environnement déterminent le seuil à partir duquel la levure s'accouple. La sous-unité régulatrice de la kinase à protéine A (PKA),Bcy1, une composante clé du réseau signalétique du senseur aux nutriments, interagit directement avec la sous-unité α des petites protéines G, Gpa1, le premier effecteur dans le réseau de réponse aux phéromones. L'interaction Bcy1-Gpa1 est accrue lorsque la cellule croit en présence d'un sucre idéal, le glucose, diminuant la concentration seuil auquel la décision d'accouplement est activée. Compromettre l'interaction Bcy1-Gpa1 ou inactiver Bcy1 accroît la concentration seuil nécessaire à une réponse aux phéromones. Nous argumentons qu'en ajustant leur sensibilité, les levures peuvent intégrer le stimulus provenant des phéromones au niveau du glucose extracellulaire, priorisant la décision de survie dans un milieu pauvre ou continuer leur cycle sexuel en choisissant un accouplement. / Evolution has resulted in numerous innovations that allow organisms to maximize their fitness by choosing particular mating partners, including secondary sexual characteristics, behavioural patterns, chemical attractants and corresponding sensory mechanisms. The haploid yeast Saccharomyces cerevisiae selects mating partners by interpreting the concentration gradient of pheromone secreted by potential mates through a network of mitogen-activated protein kinase (MAPK) signaling proteins. The mating decision in yeast is an all-or-none, or switch-like, response that allows cells to make accurate decisions about which among potential partners to mate with and to filter weak pheromone signals, thus avoiding inappropriate commitment to mating by responding only at or above critical concentrations when a mate is sufficiently close. The molecular mechanisms that govern the switch-like mating decision are poorly understood. In this thesis I demonstrate that the switching mechanism arises from competition between the MAPK Fus3 and a phosphatase Ptc1 for control of the phosphorylation state of four sites on the scaffold protein Ste5. This competition results in a switch-like dissociation of Fus3 from Ste5 that is necessary to generate the switch-like mating response. Thus, the decision to mate is made at an early stage in the pheromone pathway and occurs rapidly, perhaps to prevent the loss of the potential mate to competitors. We argue that the architecture of the Fus3–Ste5–Ptc1 circuit generates a novel ultrasensitivity mechanism that resembles “zero-order ultrasensitivity”, which is robust to variations in the concentrations of these proteins. This robustness helps assure that mating can occur despite stochastic or genetic variation between individuals. I then demonstrate that during the mating response, an early event of Ste5 recruitment to plasma membrane is ultrasensitive and that it is generated by dephosphorylation of eight N-terminal phosphosites on Ste5 by the phosphatase Ptc1 when associated with Ste5 via the polarization protein Bem1. Interference with this mechanism results in loss of ultrasensitivity and reduced amplitude and therefore fidelity of the pheromone signaling response. These changes are reflected in reduced fidelity and accuracy of the morphogenic mating response. Polarized assembly of signaling protein complexes at the plasma membrane surface is a general theme recapitulated in all organisms from bacteria to humans. Such complexes can increase the efficiency, fidelity and specificity of signal transduction. Together with our previous findings, our results demonstrate that ultrasensitivity, accuracy and robustness of the pheromone response occurs through regulation of the stoichiometry of phosphorylation of two clusters of phosphosites on Ste5, by Ptc1, one cluster mediating ultrasensitive recruitment of Ste5 to the membrane and the other, ultrasensitive dissociation and activation of the terminal MAP kinase Fus3. The role of Ste5 as a direct modulator of a cell-fate decision expands the functional repertoire of scaffold proteins beyond providing specificity and efficiency of information processing. Regulation of dynamic signal-response characteristics through such modular regulation of clusters of phosphosites may be a general means by which cell fate decisions are achieved. Similar mechanisms may govern cellular decisions in higher organisms and be disrupted in cancer. Finally, in a related theme, I present the discovery of a novel mechanisms by which the threshold of pheromone response is controlled by a nutrient-sensing pathway, thus adjusting the set-point at which the quantity and quality of nutrients available in the environment set the threshold of pheromone at which yeast will mate. The regulatory subunit of protein kinase A (PKA), Bcy1, a key component of a nutrient sensing signaling network, directly interacts with the α subunit of G-protein, Gpa1, the primary effector of the pheromone signaling network. The Bcy1-Gpa1 interaction is enhanced when cells are grown in their ideal carbon source glucose, lowering the threshold concentration at which the mating response is activated. Disruption of Bcy1-Gpa1 interaction or Bcy1 deletion increased the threshold concentration for the mating response. We argue that by adjusting their sensitivity, yeast can integrate pheromone stimulus with glucose levels and prioritize decisions to survive in a nutrient-starved environment or to continue their sexual cycle by mating.

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Temporal control, inherent in all biological processes, relies on intrinsic systems to govern periodical behaviors and physiological responses. The circadian clock, a vital timekeeper, enables organisms to anticipate and adjust to daily environmental changes. In mammals, the circadian clock is organized hierarchically, with a central master clock in the hypothalamic suprachiasmatic nucleus regulating peripheral clocks distributed across the body. To maintain coherent circadian rhythms at the tissue level, peripheral oscillators exchange intercellular coupling factors by paracrine signaling pathways to synchronize. At the individual cell scale, the circadian clock interacts with another periodical biological process: the cell cycle. However, the mechanisms governing this interplay remain poorly elucidated. Here, we explore the influence of extracellular circadian synchronization on the intracellular coordination between the circadian clock and the cell cycle. To do so, we combined a mathematical model and long-term live-imaging recordings at the single-cell and population level of a human cell line. We show that the global circadian and cell cycle coordination within individual cells is disrupted when the extracellular circadian synchronization is lost, obstructing collective tissue growth. Populations with coherent circadian rhythms display rhythmic growth oscillations, uncovering a novel global regulator of tissue dynamics. Knocking down core circadian elements abolished these effects, revealing the fundamental role of circadian clock control as a timing mechanism. These findings advance our understanding of how biological systems maintain equilibrium and regulate proliferation in normal and pathological conditions. The circadian clock plays a crucial role in orchestrating cell proliferation, impacting tumor initiation, growth, and treatment responses. Recent research has reported significant changes in drug response for different administration hours throughout the day, highlighting the benefits of aligning treatment strategies to the inherent circadian rhythm. However, chronotherapy is still omitted in clinical practice, primarily due to a lack of understanding of the underlying mechanisms driving time-dependent drug responses. Currrently, no standardized protocols exist for identifying these temporal factors. Therefore, we developed a combined mathematical and experimental approach to identify the factors influencing time-dependent drug sensitivity in human cells. Our results show how circadian and drug properties independently shape time-of-day drug responses, offering novel insights into the time-dependent treatment outcome. This framework holds potential for developing personalized treatment schedules aligned with the internal circadian clock, optimizing cancer therapeutical strategies. On the other hand, tissue growth masks heterogeneous proliferation patterns at the single-cell level, potentially jeopardizing the treatment outcome, which cannot be exclusively attributed to circadian clock regulation. Clustering the cells upon their overall number of divisions, the proliferative patterns remain strikingly constant across different tissues, a phenomenon reported by several recent studies. This consistency implies the existence of a common underlying mechanism that is currently unknown. Proliferation control relies on a set of checkpoint mechanisms that accurately and quickly detect DNA damage. The onset of cellular stress triggers the activation of the p53 protein, orchestrating the expression of hundreds of genes responsible for cell cycle regulation or apoptosis, among other functions. Here, we present evidence that changes in cellular stress levels contribute to the gradual proliferation variability. Specifically, different DNA damage levels are encoded quantitively into signal parameters of p53 and p21 proteins in a gradual manner, shaping proliferation activity proportionally. These results propose a novel function of the p53-p21 signaling network in deciphering and decoding the magnitude of DNA damage to adjust and control proliferation. / This study examines how extracellular circadian synchronization affects the coordination between the circadian clock and the cell cycle. By combining mathematical modeling and long-term live imaging of human cells, we show that loss of synchronization disrupts global circadian and cell cycle coordination in individual cells, hindering tissue growth. When circadian rhythms are coherent, rhythmic growth oscillations occur, indicating a global tissue dynamics regulator. Knocking down key circadian elements abolished these effects, emphasizing the circadian clock's timing role. These findings enhance our understanding of biological balance and proliferation regulation in both normal and pathological states. The circadian clock is also vital in cell proliferation, influencing tumor growth and treatment responses. Drug responses vary depending on the time of day, highlighting the importance of aligning treatments with circadian rhythms. However, chronotherapy is not widely used in clinical practice due to insufficient understanding of the underlying mechanisms. Our approach identifies factors affecting time-dependent drug sensitivity, offering insights into personalized treatment schedules. Tissue growth masks single-cell proliferation patterns, essential for effective treatment. By clustering cells based on division numbers, we found consistent proliferation patterns across tissues, suggesting an unknown underlying mechanism. The p53-p21 signaling network regulates proliferation by quantifying DNA damage and adjusting cell cycle responses. This study reveals how p53-p21 signaling decodes DNA damage levels to control proliferation. / Diese Studie erforscht, wie extrazelluläre zirkadiane Synchronisation die Abstimmung zwischen der zirkadianen Uhr und dem Zellzyklus beeinflusst. Durch mathematische Modellierung und langfristige Live-Bildgebung an menschlichen Zellen zeigt sie, dass der Verlust der Synchronisation die zirkadiane und Zellzykluskoordination stört und Gewebewachstum hemmt. Bei kohärenten zirkadianen Rhythmen entstehen rhythmische Wachstumsoszillationen, die eine globale Steuerung der Gewebedynamik andeuten. Das Ausschalten zirkadianer Elemente beseitigt diese Effekte und verdeutlicht die zeitliche Rolle der zirkadianen Uhr. Diese Ergebnisse liefern Einblicke in die biologische Balance und Regulierung der Proliferation in normalen und pathologischen Zuständen. Die zirkadiane Uhr beeinflusst die Zellproliferation, das Tumorwachstum und die Wirkung von Behandlungen. Arzneimittelreaktionen variieren tageszeitabhängig, was die Relevanz der Chronotherapie unterstreicht – der Anpassung von Therapien an zirkadiane Rhythmen. Allerdings wird Chronotherapie selten klinisch genutzt, da die zugrundeliegenden Mechanismen nicht ausreichend verstanden sind. Diese Studie identifiziert Faktoren, die die zeitabhängige Arzneimittelempfindlichkeit beeinflussen, und bietet Perspektiven für personalisierte Therapien. Gewebewachstum verdeckt Proliferationsmuster einzelner Zellen, die für Behandlungen entscheidend sind. Durch Clusteranalysen der Zellteilungen zeigten sich konsistente Muster in Geweben, was auf unbekannte Mechanismen hindeutet. Das p53-p21-Signalnetzwerk reguliert die Proliferation, indem es DNA-Schäden bewertet und Zellzyklusreaktionen anpasst. Die Studie zeigt, wie dieses Netzwerk DNA-Schäden interpretiert, um Zellwachstum zu steuern.