Nuclear translocation kinetics of NF-κB in macrophages challenged with pathogens in a microfluidic platform

James, Conrad D.; Moorman, Matthew W.; Carson, Bryan.; Branda, Catherine; Lantz, Jeffrey W.; Manginell, Ronald P.; Martino, Anthony; Singh, Anup K.

doi:10.1007/s10544-008-9281-5

Cited by 16 publications

(16 citation statements)

References 31 publications

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“…We and others predicted and demonstrated that responses of the NF-κB system to low TNFα doses, as well as low LPS doses, are highly stochastic, and only a fraction of cells exhibit measurable NF-κB activation [27],[34],[74],[75]. Our ELISpot data on 3T3 cells and macrophages show that only a small fraction of cells secrete TNFα.…”

Section: Methodssupporting

confidence: 53%

Spontaneous NF-κB Activation by Autocrine TNFα Signaling: A Computational Analysis

et al. 2013

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NF-κB is a key transcription factor that regulates innate immune response. Its activity is tightly controlled by numerous feedback loops, including two negative loops mediated by NF-κB inducible inhibitors, IκBα and A20, which assure oscillatory responses, and by positive feedback loops arising due to the paracrine and autocrine regulation via TNFα, IL-1 and other cytokines. We study the NF-κB system of interlinked negative and positive feedback loops, combining bifurcation analysis of the deterministic approximation with stochastic numerical modeling. Positive feedback assures the existence of limit cycle oscillations in unstimulated wild-type cells and introduces bistability in A20-deficient cells. We demonstrated that cells of significant autocrine potential, i.e., cells characterized by high secretion of TNFα and its receptor TNFR1, may exhibit sustained cytoplasmic–nuclear NF-κB oscillations which start spontaneously due to stochastic fluctuations. In A20-deficient cells even a small TNFα expression rate qualitatively influences system kinetics, leading to long-lasting NF-κB activation in response to a short-pulsed TNFα stimulation. As a consequence, cells with impaired A20 expression or increased TNFα secretion rate are expected to have elevated NF-κB activity even in the absence of stimulation. This may lead to chronic inflammation and promote cancer due to the persistent activation of antiapoptotic genes induced by NF-κB. There is growing evidence that A20 mutations correlate with several types of lymphomas and elevated TNFα secretion is characteristic of many cancers. Interestingly, A20 loss or dysfunction also leaves the organism vulnerable to septic shock and massive apoptosis triggered by the uncontrolled TNFα secretion, which at high levels overcomes the antiapoptotic action of NF-κB. It is thus tempting to speculate that some cancers of deregulated NF-κB signaling may be prone to the pathogen-induced apoptosis.

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Section: Methodssupporting

confidence: 53%

Spontaneous NF-κB Activation by Autocrine TNFα Signaling: A Computational Analysis

et al. 2013

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“…induce NF-κB to translocate from the cytoplasm to the nucleus via phosphorylation and degradation of its cytoplasmic inhibitor IκB. Such process was previously studied in microfluidic devices using fluorescence imaging when the protein of interest was tagged with a fluorescent protein marker20, 21. This translocation process has also been routinely studied by the combination of subcellular fractionation and Western blotting.…”

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confidence: 99%

One-step extraction of subcellular proteins from eukaryotic cells

et al. 2010

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Conventional biochemical analysis mainly focuses on the expression level of cellular proteins from entire cells. However, it has been increasingly acknowledged that the subcellular location of proteins often carries important information. Analysis of subcellular proteins conventionally requires subcellular fractionation which involves two steps: cell lysis to release proteins and high-speed centrifugation to separate the homogenate. Such approach requires bulky and expensive equipment and is not compatible with processing scarce cell samples of limited volume. In this study, we apply microfluidic flow-through electroporation to breach cell membranes and extract cytosolic proteins selectively in a single step. We demonstrate that this approach allows monitoring the translocation of the transcription factor NF-κB from the cytosol to the nucleus without the need of subcellular fractionation. Our technique is compatible with the processing of samples of various sizes and provides a simple and universal tool for bioanalytical analysis and spatial proteomics.

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“…In the future, we expect that more researchers will turn to classic immune cell types or cell lines that mimic the properties of immune cells, such as the macrophage like RAW 264.7 line [26]. …”

Section: Workflow Methods and Challengesmentioning

confidence: 99%

“…Instead, researchers have now begun to study the NF-κB response to different TNF-α concentrations [10] and time-varying TNF-α stimulation [9, 14, 22]. Recent interests also include the NF-κB activation dynamics under different ligands [13, 18, 23], upon drug perturbations [24, 25], and in response to live or dead bacteria [26–29]. Furthermore, multiple outputs can be observed in the same cell through time, for example the dynamics of NF-κB localization and IκBα expression [12, 30].…”

Section: Why Bother With Single Cell Imaging?mentioning

confidence: 99%

High-throughput, single-cell NF-κB dynamics

Lee¹,

Covert²

2010

Current Opinion in Genetics & Development

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SummarySingle cells in a population often respond differently to perturbations in the environment. Live-cell microscopy has enabled scientists to observe these differences at the single-cell level. Some advantages of live-cell imaging over population-based methods include better time resolution, higher sensitivity, automation, and richer datasets. One specific area where live-cell microscopy has made a significant impact is the field of NF-κB signaling dynamics, and recent efforts have focused on making live-cell imaging of these dynamics more high throughput. We highlight the major aspects of increasing throughput and describe a current system that can monitor, image and analyze the NF-κB activation of thousands of single cells in parallel.Genetically identical, individual cells in a common environment often vary significantly in their response to perturbations. The behavioral variation between single cells can be observed experimentally using live-cell microscopy, as has been demonstrated in bacterial [1,2], yeast [3], and mammalian systems [4][5][6]. One common feature of these studies is that measuring the dynamics of single cells yields new and exciting insights about phenotypic heterogeneity in a population.NF-κB is a family of transcription factors that control the expression of hundreds of genes [7]. Beyond NF-κB's importance as a key player in innate immunity [8], study of NF-κB activation as a case study in the field of systems biology has been very productive in recent years [9][10][11][12][13][14][15]. The initial computational models of NF-κB signaling demonstrated the power of combined computational and experimental analyses in understanding the NF-κB response to . Further studies continue to reveal the role of additional components [14,17], the response to other ligands [11,13], and the behavior of NF-κB in single cells [9,12,18].Live-cell microscopy has made a substantial impact on the field of NF-κB dynamics, illuminating how population responses can be interpreted as the coordinated action of single cells. These microscopy-based studies have been limited to the analysis of relatively few cells, but recent developments have increased this throughput dramatically. The purpose of this short review is to highlight some important reasons why live-cell imaging has been useful in studying NF-κB, and then to summarize the workflow, methods and challenges of NF-κB imaging with a focus on recent advances that have made this technology high-throughput. © 2010 Elsevier Ltd. All rights reserved.Correspondence to: Markus W. Covert, mcovert@stanford.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers t...

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Nuclear translocation kinetics of NF-κB in macrophages challenged with pathogens in a microfluidic platform

Cited by 16 publications

References 31 publications

Spontaneous NF-κB Activation by Autocrine TNFα Signaling: A Computational Analysis

Spontaneous NF-κB Activation by Autocrine TNFα Signaling: A Computational Analysis

One-step extraction of subcellular proteins from eukaryotic cells

High-throughput, single-cell NF-κB dynamics

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