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Regulation of Signal Transduction Studied by Quantitative Single Cell Measurements

English title Regulation of Signal Transduction Studied by Quantitative Single Cell Measurements
Applicant Pelet Serge
Number 165844
Funding scheme SNSF Professorships
Research institution Département de Microbiologie Fondamentale Faculté de Biologie et de Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Biochemistry
Start/End 01.09.2016 - 31.08.2017
Approved amount 391'244.00
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All Disciplines (3)

Discipline
Biochemistry
Cellular Biology, Cytology
Experimental Microbiology

Keywords (8)

MAPK pathways; Budding yeast; Quantitative measurements; Single cell; Microscopy; Biosensors; Signaling networks; microfluidics

Lay Summary (French)

Lead
L’analyse biochimique des cellules a permis de définir des cartes statiques d’interactions entre les protéines impliquées dans des cascades de signalisation. Pour comprendre les processus dynamiques qui ont lieu dans ces voies, les réponses de cellules uniques vont être mesurées en temps réel et de manière quantitative. La microscopie combinée à la microfluidique offrent de grandes possibilités pour générer ce type de données. Ces approches vont être utilisées pour étudier l’activation des voies MAPK dans la levure et développer des nouveaux bio-senseurs fluorescents pour mesurer en temps réel la dynamique des événements induits par l’activation de la cascade de transduction. Ces mesures vont permettre de mettre à jour des mécanismes de régulation de ces voies de signalisation qui sont conservées dans toutes les cellules eucaryotes.
Lay summary

Les cellules vivent dans des environnements qui changent sans cesse. Elles ont développé des voies de signalisation complexes pour répondre à ces changements. Une structure récurrente pour transmettre ces stimuli extra-cellulaires sont les mitogen activated protein kinases (MAPK). Cette architecture a été conservée au cours de l’évolution des levures jusqu’aux mammifères. La levure de boulanger est utilisée comme système modèle pour comprendre la régulation de ces voies de signalisation.

 

Traditionnellement, les expériences en biologie sont faites sur un grand nombre de cellules. Mais chaque cellule dans la population sera légèrement différente des autres L’observation des cellules individuelles permet de découvrir de nouvelles réponses qui sont masquées quand la population est mesurée dans son ensemble. Par exemple, l’addition de NaCl au milieu de culture des levures induit l’activation de la MAPK Hog1. Quand la réponse des cellules uniques est mesurée, nous avons pu démontrer que l’activation de la kinase était uniforme dans la population. Par contre, l’expression de protéines a été détectée dans seulement une fraction des cellules. Cette expérience démontre que la même activité de MAPK peut donner lieu à des profils d’expressions très différents dans des cellules isogéniques soumises au même stress.

 

La microscopie offre une opportunité unique d’observer la réponse de cellules individuelles. La découverte de la protéine fluorescente verte permet de suivre la localisation et l’abondance de n’importe quelle protéine. Cette technique permet de mesurer l’activité de protéines impliquées dans les voies de signalisation en temps réel dans des cellules vivantes. Un but de ce projet est de développer de nouveaux senseurs pour quantifier par microscopie la dynamique de transmission du signal. Ces bio-senseur, permettront de mesurer des activations différentes de cellule à cellule et donc d’identifier de nouveau méchanismses de régulation présent dans les voies MAPK.

 

 

Direct link to Lay Summary Last update: 01.03.2016

Responsible applicant and co-applicants

Employees

Publications

Publication
Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5
van Drogen Frank, Mishra Ranjan, Rudolf Fabian, Walczak Michal J., Lee Sung Sik, Reiter Wolfgang, Hegemann Björn, Pelet Serge, Dohnal Ilse, Binolfi Andres, Yudina Zinaida, Selenko Philipp, Wider Gerhard, Ammerer Gustav, Peter Matthias (2019), Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5, in The Journal of Cell Biology, 218(9), 3117-3133.
Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability
Coutelier Héloïse, Xu Zhou, Morisse Mony Chenda, Lhuillier-Akakpo Maoussi, Pelet Serge, Charvin Gilles, Dubrana Karine, Teixeira Maria Teresa (2018), Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability, in Genes & Development, 32(23-24), 1499-1513.
Timing of gene expression in a cell‐fate decision system
Aymoz Delphine, Solé Carme, Pierre Jean‐Jerrold, Schmitt Marta, de Nadal Eulàlia, Posas Francesc, Pelet Serge (2018), Timing of gene expression in a cell‐fate decision system, in Molecular Systems Biology, 14(4), e8024.
Dynamics of signal transduction in single cells quantified by microscopy
Pelet Serge (2017), Dynamics of signal transduction in single cells quantified by microscopy, in Nielsen Jens , Hohman Stefan (ed.), Wiley, Germany, 289.
Nuclear relocation of Kss1 contributes to the specificity of the mating response.
Pelet Serge (2017), Nuclear relocation of Kss1 contributes to the specificity of the mating response., in Scientific reports, 1.
Relocation sensors to quantify signaling dynamics in live single cells.
Pelet Serge (2017), Relocation sensors to quantify signaling dynamics in live single cells., in Current opinion in biotechnology, 45, 51-58.
Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element.
Pelet Serge (2016), Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element., in PNAS, E3375–E338.
New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast
Wosika Victoria, Durandau Eric, Varidel Clémence, Aymoz Delphine, Schmitt Marta, Pelet Serge (2016), New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast, in Molecular Genetics and Genomics, 2231.
Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters
Aymoz Delphine, Wosika Victoria, Durandau Eric, Pelet Serge (2016), Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters, in Nature Communications, 7, 11304-11304.

Datasets

Timing of gene expression in a cell‐fate decision system

Author Aymoz, Delphine; Pelet, Serge
Publication date 29.03.2018
Persistent Identifier (PID) https://doi.org/10.17867/10000114
Repository IDR
Abstract
During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating‐responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contributes to determine the kinetics of transcription in a simplified cell‐fate decision system.

Collaboration

Group / person Country
Types of collaboration
Felix Naef Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Robbie Loewith /Geneva Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
David Shore / Geneva Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Teresa Teixeira France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Gian Paolo Dotto / Lausanne Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Francesc Posas and Eulalia de Nadal / Universitat Pompeu Fabra Spain (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
CIG Symposium 2017 Talk given at a conference Timeline of gene expression in budding yeast mating, quantified by dynamic translocation reporters 15.06.2017 Lausanne, Switzerland Aymoz Delphine;
Cellular Dynamics and Models Talk given at a conference Timing of gene expression in a cell fate decision system 11.04.2017 Cold Spring Harbor, United States of America Pelet Serge;
Ecole Normale Supérieure Individual talk Cell fate decisions in a yeast MAPK pathway 06.03.2017 Lyon, France Pelet Serge;
Lausanne Genomics Day Poster Real-Time quantification of gene expression in single cell using dynamic Protein Synthesis Translocation Reporters  09.02.2017 Lausanne, Switzerland Aymoz Delphine;
LS2 Annual Meeting Talk given at a conference Dynamic measurement of kinase activity in live single cell  02.02.2017 Zürich, Switzerland Durandau Eric;
18th EMBL PhD Symposium, Life by Numbers Towards Quantitative Biology Talk given at a conference Dynamic measurement of kinase activity in live single cell 17.11.2016 Heidelberg, Germany Durandau Eric;


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Mystères de l'UNIL Western Switzerland 2017

Associated projects

Number Title Start Funding scheme
172900 Regulation of Signal Transduction Studied by Quantitative Single Cell Measurements 01.09.2017 SNSF Professorships
139121 Regulation of Signal Transduction Studied by Quantitative Single Cell Measurements 01.09.2012 SNSF Professorships

Abstract

Living cells respond to changes in their environment by activating signal transduction cascades that mount an appropriate cellular response. Mitogen Activated Protein Kinase (MAPK) pathways play a central role in all eukaryotes by transducing extra-cellular cues and allowing cells to respond to stresses or growth promoting signals. Mis-regulation of these pathways in mammalian cells can lead to multiple diseases, including inflammatory response and cancer. S. cerevisiae has been used for a long time as a model system to understand the fundamental organization of these signaling cascades. Thanks to decades of genetics and biochemical analyses, the major components of these pathways have been identified and a static map of their interactions has been established. The challenge for the coming years is to obtain an understanding of the dynamic flow of information in this network.In order to achieve this, we need to acquire quantitative measurements of signaling activity in real time. Moreover, these measurements have to be performed at the single cell level, because population averaging can mask informative behaviors such as oscillation or bimodality. Microscopy is ideally suited to generate these data, if assays can be established to monitor in living cells the dynamics of signaling activity.Therefore, in the first years of this grant, my group has established novel biosensors to measure kinase activity and protein expression in the yeast MAPK pathways. These reporters have allowed us to uncover unexpected behaviors in the signal transduction. For example, by combining a kinase activity and a dynamic expression reporter system in the same cells, we have been able to monitor the interplay between kinase and transcriptional activity upon mating pheromone stimulation. We have observed a discrepancy between the fast activation of the MAPK and a delayed induction of protein expression. These data strongly suggest the implication of an unknown additional player that orchestrates the expression of target genes in coordination with the MAPK.The prolongation of this grant will focus on three major aims:Development of biosensorsWe plan to improve the sensitivity of the current bio-reporters by generating local kinase activity measurements. The palette of the available biosensors will be expanded to monitor other signaling pathways and apply them to mammalian cells.Investigation of yeast MAPK signal transductionWe will use these novel biosensors to monitor the heterogeneity in kinase activity under basal condition and upon acute stimulation of these pathways. We also plan to improve our understanding of the specificity and cross-talk in the yeast MAPK network by combing multiple sensors in the same cell. Study of the control of MAPK protein expressionWe will further study the interplay between signaling activity and protein expression output. Ultimately, we would like to understand how the promoter architecture controls the dynamics and noise of protein expression.Overall, this project will contribute to improve our understanding of the basic principles contributing to the fidelity and robustness of signal transduction in MAPK pathways. Moreover, the tools that we are developing will be made available to the entire research community such that they can be applied to solve a wide diversity of research questions in various organisms.
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