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High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations

NF-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 biol...

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Autores principales: Zambrano, Samuel, Bianchi, Marco E., Agresti, Alessandra
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942427/
https://www.ncbi.nlm.nih.gov/pubmed/24595030
http://dx.doi.org/10.1371/journal.pone.0090104
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author Zambrano, Samuel
Bianchi, Marco E.
Agresti, Alessandra
author_facet Zambrano, Samuel
Bianchi, Marco E.
Agresti, Alessandra
author_sort Zambrano, Samuel
collection PubMed
description NF-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.
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spelling pubmed-39424272014-03-06 High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations Zambrano, Samuel Bianchi, Marco E. Agresti, Alessandra PLoS One Research Article NF-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. Public Library of Science 2014-03-04 /pmc/articles/PMC3942427/ /pubmed/24595030 http://dx.doi.org/10.1371/journal.pone.0090104 Text en © 2014 Zambrano et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Zambrano, Samuel
Bianchi, Marco E.
Agresti, Alessandra
High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations
title High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations
title_full High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations
title_fullStr High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations
title_full_unstemmed High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations
title_short High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations
title_sort high-throughput analysis of nf-κb dynamics in single cells reveals basal nuclear localization of nf-κb and spontaneous activation of oscillations
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942427/
https://www.ncbi.nlm.nih.gov/pubmed/24595030
http://dx.doi.org/10.1371/journal.pone.0090104
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