Vol 9: High-Throughput Analysis of NF-B Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-B and Spontaneous Activation of Oscillations.Report as inadecuate



 Vol 9: High-Throughput Analysis of NF-B Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-B and Spontaneous Activation of Oscillations.


Vol 9: High-Throughput Analysis of NF-B Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-B and Spontaneous Activation of Oscillations. - Download this document for free, or read online. Document in PDF available to download.

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This article is from PLoS ONE, volume 9.AbstractNF-κB is a transcription factor that upon activation undergoes cycles of cytoplasmic-to-nuclear and nuclear-to-cytoplasmic transport, giving rise to so called -oscillations-. In turn, oscillations tune the transcriptional output. Since a detailed understanding of oscillations requires a systems biology approach, we developed a method to acquire and analyze large volumes of data on NF-κB dynamics in single cells. We measured the time evolution of the nuclear to total ratio of GFP-p65 in knock-in mouse embryonic fibroblasts using time-lapse imaging. We automatically produced a precise segmentation of nucleus and cytoplasm based on an accurate estimation of the signal and image background. Finally, we defined a set of quantifiers that describe the oscillatory dynamics, which are internally normalized and can be used to compare data recorded by different labs. Using our method, we analyzed NF-κB dynamics in over 2000 cells exposed to different concentrations of TNF- α α. We reproduced known features of the NF-κB system, such as the heterogeneity of the response in the cell population upon stimulation and we confirmed that a fraction of the responding cells does not oscillate. We also unveiled important features: the second and third oscillatory peaks were often comparable to the first one, a basal amount of nuclear NF-κB could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showed spontaneous random activation of the NF-κB system. Our work lays the ground for systematic, high-throughput, and unbiased analysis of the dynamics of transcription factors that can shuttle between the nucleus and other cell compartments.



Author: Zambrano, Samuel; Bianchi, Marco E.; Agresti, Alessandra

Source: https://archive.org/







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