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 sy...
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0090104&type=printable |
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| author | Samuel Zambrano Marco E Bianchi Alessandra Agresti |
| author_facet | Samuel Zambrano Marco E Bianchi Alessandra Agresti |
| author_sort | Samuel Zambrano |
| collection | DOAJ |
| 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. |
| format | Article |
| id | doaj-art-731f4dd612764e9fb752ab433e46b2d8 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj-art-731f4dd612764e9fb752ab433e46b2d82025-08-20T03:01:14ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0193e9010410.1371/journal.pone.0090104High-throughput analysis of NF-κB dynamics in single cells reveals basal nuclear localization of NF-κB and spontaneous activation of oscillations.Samuel ZambranoMarco E BianchiAlessandra AgrestiNF-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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0090104&type=printable |
| spellingShingle | Samuel Zambrano Marco E Bianchi Alessandra Agresti High-throughput analysis of NF-κB dynamics in single cells reveals basal nuclear localization of NF-κB and spontaneous activation of oscillations. PLoS ONE |
| 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 |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0090104&type=printable |
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