Abstract
BackgroundMitogen 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.ResultsWe 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0163-z) contains supplementary material, which is available to authorized users.
Highlights
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
Sensor design Nuclear localization sequences (NLSs) consist of a stretch of positively charged residues associated with importin, which will subsequently shuttle its cargo into the nucleus [14]
They observed that the NLS for the Swi6 transcription cofactor was led by a serine, which is a potential phosphorylation target
Summary
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. Cells perceive changes in their environment and integrate these extra-cellular signals with intra-cellular cues to mount an appropriate response. This is achieved by signal transduction cascades, where receptors at the plasma membrane sense the surrounding medium and transmit this information to intracellular enzymes that modulate the activity of other proteins via post-translational modifications to induce a specific response. The mitogen activated protein kinase (MAPK) pathways are a conserved family of signaling cascades which respond to a wide range of signals such as growth hormones, nutrient status, or stresses [1]. This kinase further activates two MAPKs, Fus and Kss, both of which contribute to the mating response [3, 4]
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