80 research outputs found
Molecular mechanisms for a switch-like mating decision in Saccharomyces cerevisiae
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
Utilization of the Mating Scaffold Protein in the Evolution of a New Signal Transduction Pathway for Biofilm Development
Among the hemiascomycetes, only Candida albicans must switch from the white phenotype to the opaque phenotype to mate. In the recent evolution of this transition, mating-incompetent white cells acquired a unique response to mating pheromone, resulting in the formation of a white cell biofilm that facilitates mating. All of the upstream components of the white cell response pathway so far analyzed have been shown to be derived from the ancestral pathway involved in mating, except for the mitogen-activated protein (MAP) kinase scaffold protein, which had not been identified. Here, through binding and mutational studies, it is demonstrated that in both the opaque and the white cell pheromone responses, Cst5 is the scaffold protein, supporting the evolutionary scenario proposed. Although Cst5 plays the same role in tethering the MAP kinases as Ste5 does in Saccharomyces cerevisiae, Cst5 is approximately one-third the size and has only one rather than four phosphorylation sites involved in activation and cytoplasmic relocalization
Digital signaling decouples activation probability and population heterogeneity
Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.ISSN:2050-084
Scaffold-mediated Nucleation of Protein Signaling Complexes: Elementary Principles
Proteins with multiple binding sites play important roles in cell signaling
systems by nucleating protein complexes in which, for example, enzymes and
substrates are co-localized. Proteins that specialize in this function are
called by a variety names, including adapter, linker and scaffold.
Scaffold-mediated nucleation of protein complexes can be either constitutive or
induced. Induced nucleation is commonly mediated by a docking site on a
scaffold that is activated by phosphorylation. Here, by considering minimalist
mathematical models, which recapitulate scaffold effects seen in more
mechanistically detailed models, we obtain analytical and numerical results
that provide insights into scaffold function. These results elucidate how
recruitment of a pair of ligands to a scaffold depends on the concentrations of
the ligands, on the binding constants for ligand-scaffold interactions, on
binding cooperativity, and on the milieu of the scaffold, as ligand recruitment
is affected by competitive ligands and decoy receptors. For the case of a
bivalent scaffold, we obtain an expression for the unique scaffold
concentration that maximally recruits a pair of monovalent ligands. Through
simulations, we demonstrate that a bivalent scaffold can nucleate distinct sets
of ligands to equivalent extents when the scaffold is present at different
concentrations. Thus, the function of a scaffold can potentially change
qualitatively with a change in copy number. We also demonstrate how a scaffold
can change the catalytic efficiency of an enzyme and the sensitivity of the
rate of reaction to substrate concentration. The results presented here should
be useful for understanding scaffold function and for engineering scaffolds to
have desired properties.Comment: 12 pages, 8 figure
Ultrasensitivity of the Bacillus subtilis sporulation decision
Starving Bacillus subtilis cells execute a gene expression program
resulting in the formation of stress-resistant spores. Sporulation
master regulator, Spo0A, is activated by a phosphorelay and controls
the expression of a multitude of genes, including the forespore-
specific sigma factor σF and the mother cell-specific sigma
factor σE. Identification of the system-level mechanism of the sporulation
decision is hindered by a lack of direct control over Spo0A
activity. This limitation can be overcome by using a synthetic system
in which Spo0A activation is controlled by inducing expression
of phosphorelay kinase KinA. This induction results in a switch-like
increase in the number of sporulating cells at a threshold of KinA.
Using a combination of mathematical modeling and single-cell microscopy,
we investigate the origin and physiological significance
of this ultrasensitive threshold. The results indicate that the phosphorelay
is unable to achieve a sufficiently fast and ultrasensitive
response via its positive feedback architecture, suggesting that the
sporulation decision is made downstream. In contrast, activation
of σF in the forespore and of σE in the mother cell compartments
occurs via a cascade of coherent feed-forward loops, and thereby
can produce fast and ultrasensitive responses as a result of KinA
induction. Unlike σF activation, σE activation in the mother cell
compartment only occurs above the KinA threshold, resulting in
completion of sporulation. Thus, ultrasensitive σE activation explains
the KinA threshold for sporulation induction. We therefore infer
that under uncertain conditions, cells initiate sporulation but postpone
making the sporulation decision to average stochastic fluctuations
and to achieve a robust population response
Reconstruction of complex single-cell trajectories using CellRouter
A better understanding of the cell-fate transitions that occur in complex cellular ecosystems in normal development and disease could inform cell engineering efforts and lead to improved therapies. However, a major challenge is to simultaneously identify new cell states, and their transitions, to elucidate the gene expression dynamics governing cell-type diversification. Here, we present CellRouter, a multifaceted single-cell analysis platform that identifies complex cell-state transition trajectories by using flow networks to explore the subpopulation structure of multi-dimensional, single-cell omics data. We demonstrate its versatility by applying CellRouter to single-cell RNA sequencing data sets to reconstruct cell-state transition trajectories during hematopoietic stem and progenitor cell (HSPC) differentiation to the erythroid, myeloid and lymphoid lineages, as well as during re-specification of cell identity by cellular reprogramming of monocytes and B-cells to HSPCs. CellRouter opens previously undescribed paths for in-depth characterization of complex cellular ecosystems and establishment of enhanced cell engineering approaches
PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades
G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3′-5′-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:βγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades
A Novel Genetic Screen Implicates Elm1 in the Inactivation of the Yeast Transcription Factor SBF
BACKGROUND: Despite extensive large scale analyses of expression and protein-protein interactions (PPI) in the model organism Saccharomyces cerevisiae, over a thousand yeast genes remain uncharacterized. We have developed a novel strategy in yeast that directly combines genetics with proteomics in the same screen to assign function to proteins based on the observation of genetic perturbations of sentinel protein interactions (GePPI). As proof of principle of the GePPI screen, we applied it to identify proteins involved in the regulation of an important yeast cell cycle transcription factor, SBF that activates gene expression during G1 and S phase. METHODOLOGY/PRINCIPLE FINDINGS: The principle of GePPI is that if a protein is involved in a pathway of interest, deletion of the corresponding gene will result in perturbation of sentinel PPIs that report on the activity of the pathway. We created a fluorescent protein-fragment complementation assay (PCA) to detect the interaction between Cdc28 and Swi4, which leads to the inactivation of SBF. The PCA signal was quantified by microscopy and image analysis in deletion strains corresponding to 25 candidate genes that are periodically expressed during the cell cycle and are substrates of Cdc28. We showed that the serine-threonine kinase Elm1 plays a role in the inactivation of SBF and that phosphorylation of Elm1 by Cdc28 may be a mechanism to inactivate Elm1 upon completion of mitosis. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that GePPI is an effective strategy to directly link proteins of known or unknown function to a specific biological pathway of interest. The ease in generating PCA assays for any protein interaction and the availability of the yeast deletion strain collection allows GePPI to be applied to any cellular network. In addition, the high degree of conservation between yeast and mammalian proteins and pathways suggest GePPI could be used to generate insight into human disease
Reversible and Noisy Progression towards a Commitment Point Enables Adaptable and Reliable Cellular Decision-Making
Cells must make reliable decisions under fluctuating extracellular conditions, but also be flexible enough to adapt to such changes. How cells reconcile these seemingly contradictory requirements through the dynamics of cellular decision-making is poorly understood. To study this issue we quantitatively measured gene expression and protein localization in single cells of the model organism Bacillus subtilis during the progression to spore formation. We found that sporulation proceeded through noisy and reversible steps towards an irreversible, all-or-none commitment point. Specifically, we observed cell-autonomous and spontaneous bursts of gene expression and transient protein localization events during sporulation. Based on these measurements we developed mathematical population models to investigate how the degree of reversibility affects cellular decision-making. In particular, we evaluated the effect of reversibility on the 1) reliability in the progression to sporulation, and 2) adaptability under changing extracellular stress conditions. Results show that reversible progression allows cells to remain responsive to long-term environmental fluctuations. In contrast, the irreversible commitment point supports reliable execution of cell fate choice that is robust against short-term reductions in stress. This combination of opposite dynamic behaviors (reversible and irreversible) thus maximizes both adaptable and reliable decision-making over a broad range of changes in environmental conditions. These results suggest that decision-making systems might employ a general hybrid strategy to cope with unpredictably fluctuating environmental conditions
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