BACKGROUND: The 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. RESULTS: To 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. CONCLUSIONS: Taken 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.
Bartfeld, S., Hess, S., Bauer, B., Machuy, N., Ogilvie, L. A., Schuchhardt, J., & Meyer, T. F. (2010). High-throughput and single-cell imaging of NF-κB oscillations using monoclonal cell lines. BMC Cell Biology, 11. https://doi.org/10.1186/1471-2121-11-21