Dataset Information

High-throughput analysis of NF-?B dynamics in single cells reveals basal nuclear localization of NF-?B and spontaneous activation of oscillations.

ABSTRACT: NF-?B 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-?B 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-?B dynamics in over 2000 cells exposed to different concentrations of TNF- ? ?. We reproduced known features of the NF-?B 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-?B could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showed spontaneous random activation of the NF-?B 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.

SUBMITTER: Zambrano S

PROVIDER: S-EPMC3942427 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

High-throughput analysis of NF-κB dynamics in single cells reveals basal nuclear localization of NF-κB and spontaneous activation of oscillations.

Zambrano Samuel S Bianchi Marco E ME Agresti Alessandra A

PloS one 20140304 3

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 ...[more]

PMID: 24595030

Similar Datasets

Project description:BackgroundThe nuclear factor-kappaB (NF-kappaB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-kappaB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-kappaB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-kappaB-induced genes is regulated by differential dynamics of single NF-kappaB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-kappaB activation have been observed in response to the cytokine tumor necrosis factor alpha (TNFalpha), little is known about the occurrence of oscillations in response to bacterial infections.ResultsTo quantitatively assess NF-kappaB dynamics we generated human and murine monoclonal cell lines that stably express the NF-kappaB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-kappaB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed.ConclusionsTaken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-kappaB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-kappaB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions.

Dataset Information

High-throughput analysis of NF-?B dynamics in single cells reveals basal nuclear localization of NF-?B and spontaneous activation of oscillations.

Publications

High-throughput analysis of NF-κB dynamics in single cells reveals basal nuclear localization of NF-κB and spontaneous activation of oscillations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets