title: High-throughput analysis of NF-κB dynamics in single cells reveals basal nuclear localization of NF-κB and spontaneous activation of oscillations.
abstract: 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.
authors.affiliation: San Raffaele University, Milan, Italy.
authors.forename: Samuel
authors.initials: S
authors.lastname: Zambrano
authors.affiliation: San Raffaele University, Milan, Italy; San Raffaele Scientific Institute, Genetics and Cell Biology Division, Milan, Italy.
authors.forename: Marco E
authors.initials: ME
authors.lastname: Bianchi
authors.affiliation: San Raffaele Scientific Institute, Genetics and Cell Biology Division, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Agresti
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D016000:Cluster Analysis; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D041681:NIH 3T3 Cells; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D059010:Single-Cell Analysis; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016328:NF-kappa B; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins