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          Institute: MPI für Infektionsbiologie     Collection: Department of Molecular Biology     Display Documents



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ID: 533652.0, MPI für Infektionsbiologie / Department of Molecular Biology
High-throughput and single-cell imaging of NF-kappa B oscillations using monoclonal cell lines
Authors:Bartfeld, Sina; Hess, Simone; Bauer, Bianca; Machuy, Nikolaus; Ogilvie, Lesley A.; Schuchhardt, Johannes; Meyer, Thomas F.
Language:English
Date of Publication (YYYY-MM-DD):2010-03-16
Title of Journal:BMC Cell Biology
Journal Abbrev.:BMC Cell Biol.
Volume:11
Sequence Number of Article:21
Copyright:© 2010 Bartfeld et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Review Status:Peer-review
Audience:Experts Only
Abstract / Description:Background: The nuclear factor-kappa B (NF-kappa B) 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-kappa B system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-kappa B activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-kappa B-induced genes is regulated by differential dynamics of single NF-kappa B subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-kappa B activation have been observed in response to the cytokine tumor necrosis factor alpha (TNF alpha), little is known about the occurrence of oscillations in response to bacterial infections. Results: To quantitatively assess NF-kappa B dynamics we generated human and murine monoclonal cell lines that stably express the NF-kappa B 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-kappa B 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-kappa B kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-kappa B 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.
Comment of the Author/Creator:This work was supported by funding under the Sixth Research Framework Programme of the European Union, Project INCA (LSHC-CT-2005-018704) and by the BMBF through the RNAi-Net (Grant No. 0313938A) to TFM.
External Publication Status:published
Document Type:Article
Communicated by:Hilmar Fünning
Affiliations:MPI für Infektionsbiologie/Department of Molecular Biology
External Affiliations:MicroDiscovery GmbH, Berlin, Germany.; Hannover Med Sch MHH, D-30625 Hannover, Germany.
Identifiers:ISI:000276333600001 [ID No:1]
ISSN:1471-2121 [ID No:2]
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