Signaling systems regulate the flow of cellular information by organizing proteins in space and time to coordinate a variety of cellular activities that are critical for the proper development, function, and maintenance of cells. Signaling molecules can exhibit several levels of complexity through the utilization of modular protein interactions, which can generate simple linear behaviors or complex behaviors such as ultrasensitivity. Protein modularity also serves as the basis for the vast protein networks that form the regulatory networks that govern several biological activities. My work focuses on the importance of protein modularity in complex biological systems, in particular the regulatory pathways of spindle positioning. The first part of my work involves the construction of a synthetic regulatory network using modular protein interactions in an effort to understand the complex behavior of the natural spindle orientation regulator Pins. Utilizing well-characterized protein domains and their binding partners, I built an autoinhibited protein switch that can be activated by a small protein domain. We found that the input-output relationship of the synthetic protein switch could be tuned by the simple addition of "decoy" domains, domains that bind and sequester input signal, thereby impeding the onset of the output response to generate an input threshold. By varying the number and affinities of the decoy domains, we found that we could transform a simple linear response into a complex, ultrasensitive one. Thus, modular protein interactions can serve as a source of complex behaviors. The second part of my work focuses on elucidating the molecular mechanisms underlying spindle positioning in the Drosophila neuroblast. I found that Pins orients the mitotic spindle by coordinating two opposite-polarity microtubule motors Dynein and Kinesin-73 through its multiple domains. Kinesin-73 also relies on its modular domain architecture to perform its duties in Pins-mediated spindle positioning, where its N-terminal half functions in coordinating cortical-microtubule capture while its C-terminal half functions as a region necessary for the activation of Dynein. Thus, modular protein design allows for the organization of spindle orientation regulators in space to achieve the complex biological activity that is spindle positioning. This dissertation includes previously published and unpublished coauthored material. / 10000-01-01

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.

The ability of cells to react to changes in their environment is critical to their survival. Effective decision making strategies leading to the activation of specific intracellular pathways hinge on cells sensing and processing extracellular variation. We will only be able to understand and manipulate how cells make decisions if we understand the “design” of the mechanisms that enable them to make such decisions, in terms of how they function, and in terms of their limitations and architecture. In this thesis, using mathematical modelling, I revisited classical signal sensing and transduction mechanisms in light of recent developments in methodological approaches and data collection. I studied the sensing characteristics of one of the simplest of sensors, the allosteric sensor, to understand the limits and effectiveness of its “design”. Using the classical Monod-Wyman-Changeux model of allostery, I defined and evaluated six engineering-inspired characteristics as a function of the parameters and number of sensors. I found that specifying one characteristic strongly constrains others and I determined the trade-offs that follow from these constraints. I also calculated the probability distribution of the number of input molecules that maximizes information transfer and, as a consequence, the number of environmental states a given population of sensors can discriminate between. Next, I proposed a new general model of phosphorylation cycles that can account for experimental reports of ultrasensitivity occurring in regimes that are far away from Goldbeter and Koshland’s zero-order saturation, the classical ultrasensitivity-generating mechanism. The new model exhibits robust ultrasensitivity in “anti-zero-order” regimes. The degree of ultrasensitivity, its limits, and its dependence on the parameters of the system are analytically tractable. The model can, additionally, explain in an intuitive way some puzzling experimental observations. Finally, I addressed the problem of integrating different types of signals from multiple sources, and performed some preliminary exploration of how cells can “learn” to associate and dissociate correlated signals in non-evolutionary time-scales. This work provides insights into the function and robustness of signal sensing and transduction mechanisms and as such is applicable to both the study of endogenous systems and the design of synthetic ones.

xiv, 81 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Protein-protein interaction networks translate environmental inputs into specific physiological outputs. The signaling proteins in these networks require regulatory mechanisms to ensure proper molecular function. Two common regulatory features of signaling proteins are autoinhibition and ultrasensitivity. Autoinhibition locks the protein in an inactive state through cis interactions with a regulatory module until it is activated by a specific input signal. Ultrasensitivity, defined as steep activation after a threshold, allows cells to convert graded inputs into more switch-like outputs and can lead to complex decision making behaviors such as bistability. Although these mechanisms are common features of signaling proteins, their molecular origins are poorly understood. I used the Drosophila Pins protein, a regulator of spindle positioning in neuroblast cells, as a model to study the molecular origin and function of autoinhibition and ultrasensitivity. Pins and its binding partners. Gαi and Mud, form a signaling pathway required for coordinating spindle positioning with cellular polarity in Drosophila neuroblasts. I found Pins switches from an autoinhibited to an activate state by modular allostery. Gαi binding to the third of three GoLoco (GL) domains allows Pins to interact with the microtubule binding protein Mud. The GL3 region is required for autoinhibitoon, as amino acids upstream and within GL3 constitute this regulatory behavior. This autoinhibitory module is conserved in LGN, the mammalian Pins orthologue. I also demonstrated that Gαi activation of Pins is ultrasensitive. A Pins protein containing inactivating point mutations to GLs l and 2 exhibits non-ultrasensitive (graded) activation. Ultrasensitivity is required for Pins function in vivo as the graded Pins mutant fails to robustly orient the mitotic spindle. I considered two models for the source of ultrasensitivity in this pathway: cooperative or "decoy" Gai binding. I found ultrasensitivity arises from a decoy mechanism in which GLs 1 and 2 compete with the activating GL3 for the input, Gai. These findings suggest that molecular ultrasensitivity can be generated without cooperativity. This decoy mechanism is relatively simple, suggesting ultrasensitive responses can be evolved by the inclusion of domain repeats, a common feature observed in signaling proteins. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Tom Stevens, Chairperson, Chemistry; Kenneth Prehoda, Member, Chemistry; Christopher Doe, Member, Biology; Peter von Hippel, Member, Chemistry; Karen Guillemin, Outside Member, Biology

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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