Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

Détails

Ressource 1Télécharger: s12915-015-0163-z.pdf (1868.79 [Ko])
Etat: Serval
Version: Final published version
ID Serval
serval:BIB_8B514691F3BB
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.
Périodique
BMC Biology
Auteur(s)
Durandau E., Aymoz D., Pelet S.
ISSN
1741-7007 (Electronic)
ISSN-L
1741-7007
Statut éditorial
Publié
Date de publication
2015
Peer-reviewed
Oui
Volume
13
Pages
55
Langue
anglais
Résumé
BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades.
RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage.
CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.
Mots-clé
MAP Kinase Signaling System, Microscopy, Fluorescence/methods, Mitogen-Activated Protein Kinases/metabolism, Phosphorylation, Saccharomyces cerevisiae/cytology, Saccharomyces cerevisiae/metabolism, Saccharomyces cerevisiae Proteins/metabolism, Single-Cell Analysis/methods
Pubmed
Web of science
Open Access
Oui
Création de la notice
27/08/2015 10:34
Dernière modification de la notice
08/05/2019 21:42
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