%0 Journal Article
%T High-Throughput Analysis of NF-百B Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-百B and Spontaneous Activation of Oscillations
%A Samuel Zambrano
%A Marco E. Bianchi
%A Alessandra Agresti
%J PLOS ONE
%D 2014
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0090104
%X NF-百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-百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-百B dynamics in over 2000 cells exposed to different concentrations of TNF- 汐 汐. We reproduced known features of the NF-百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-百B could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showed spontaneous random activation of the NF-百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.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0090104