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

NF-kB is a transcription factor that upon activation undergoes cycles of cytoplasmic-to-nuclear and nuclear-to-cytoplasmictransport, giving rise to so called ‘‘oscillations’’. In turn, oscillations tune the transcriptional output. Since a detailedunderstanding of oscillations requires a systems biology approach, we developed a method to acquire and analyze largevolumes of data on NF-kB dynamics in single cells. We measured the time evolution of the nuclear to total ratio of GFP-p65in knock-in mouse embryonic fibroblasts using time-lapse imaging. We automatically produced a precise segmentation ofnucleus and cytoplasm based on an accurate estimation of the signal and image background. Finally, we defined a set ofquantifiers that describe the oscillatory dynamics, which are internally normalized and can be used to compare datarecorded by different labs. Using our method, we analyzed NF-kB dynamics in over 2000 cells exposed to differentconcentrations of TNF- a a. We reproduced known features of the NF-kB system, such as the heterogeneity of the responsein the cell population upon stimulation and we confirmed that a fraction of the responding cells does not oscillate. We alsounveiled important features: the second and third oscillatory peaks were often comparable to the first one, a basal amountof nuclear NF-kB could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showedspontaneous random activation of the NF-kB system. Our work lays the ground for systematic, high-throughput, andunbiased analysis of the dynamics of transcription factors that can shuttle between the nucleus and other cellcompartments.