Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.

The Nuclear Factor kappa B (NF-κB) transcription factor regulates cellular stress responses and the immune response to infection. NF-κB activation results in oscillations in nuclear NF-κB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor alpha (TNFα) at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals analyzed, we observed synchronous cycles of NF-κB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses, gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-κB-dependent gene expression, supporting a functional role for oscillation frequency.Eukaryotic cells interpret multiple signals to coordinate the activity of transcription factors, which modulate the expression of target genes. NF-κB signaling in many mammalian cell types regulates responses to pathogens and stresses (1). NF-κB, most commonly comprising a dimer of RelA and p50, is bound in the cytoplasm of unstimulated cells by inhibitor kappa B (IκB) proteins. Stimulation by cytokines such as TNFα activates the inhibitor kappa B kinase (IKK) complex that phosphorylates IκB proteins, leading to IκB degradation and NF-κB translocation into the nucleus. Activated NF-κB regulates transcription from promoter regions of approximately 300 genes, including those encoding cytokines and several NF-κB family members which can feedback to regulate the system (2). Signaling through NF-κB can regulate diverse cellular outcomes including cell death or division (3). How such a diversity of responses is generated has remained unclear.Real-time fluorescence imaging and mathematical modeling have shown that the activity of the NF-κB system can be oscillatory (4). This raised the possibility that, as with calcium (5) (7)). In contrast to the conclusions of other reports (9, 10), we observed oscillations in the translocation of RelA-dsRedxp fusion protein in single transiently transfected MEFs (Fig. 1, C, D and E). These data (as well as bulk cell chromatin immunoprecipitation (ChIP) assays, Fig. S1), suggest that oscillations are a normal response to TNFα stimulation.In an inflammatory tissue, cells receive pulsatile signals such as TNFα from neighboring cells (11,12). To mimic this, we exposed cells to 5 min pulses of TNFα at various intervals, followed by wash-off. When stimulated at 200-min intervals, RelA-dsRedxp fusion protein expressed in SK-N-AS cells showed synchronous translocations from the cytoplasm to the nucleus and back of equal magnitude in response to each successive pulse (Fig. 2, A and B and fig. S4). In contrast, whereas stimulation at 100 or 60 min intervals also caused synchronous cell responses, there was significant reduction in the magnitude ...

Cycling of gene expression in individual cells is controlled by dynamic chromatin remodeling.

Populations of cells are almost always heterogeneous in function and fate. To understand the plasticity of cells, it is vital to measure quantitatively and dynamically the molecular processes that underlie cell-fate decisions in single cells. Early events in cell signalling often occur within seconds of the stimulus, whereas intracellular signalling processes and transcriptional changes can take minutes or hours. By contrast, cell-fate decisions, such as whether a cell divides, differentiates or dies, can take many hours or days. Multiparameter experimental and computational methods that integrate quantitative measurement and mathematical simulation of these noisy and complex processes are required to understand the highly dynamic mechanisms that control cell plasticity and fate.

SummaryControlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the ®lamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to b-glucuronidase (GUS), luciferase (LUC) and green uorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

Heterogeneity between individual cells is a common feature of dynamic cellular processes, including signaling, transcription, and cell fate; yet the overall tissue level physiological phenotype needs to be carefully controlled to avoid fluctuations. Here we show that in the NF-κB signaling system, the precise timing of a dual-delayed negative feedback motif [involving stochastic transcription of inhibitor κB (IκB)-α and -ε] is optimized to induce heterogeneous timing of NF-κB oscillations between individual cells. We suggest that this dual-delayed negative feedback motif enables NF-κB signaling to generate robust single cell oscillations by reducing sensitivity to key parameter perturbations. Simultaneously, enhanced cell heterogeneity may represent a mechanism that controls the overall coordination and stability of cell population responses by decreasing temporal fluctuations of paracrine signaling. It has often been thought that dynamic biological systems may have evolved to maximize robustness through cell-to-cell coordination and homogeneity. Our analyses suggest in contrast, that this cellular variation might be advantageous and subject to evolutionary selection. Alternative types of therapy could perhaps be designed to modulate this cellular heterogeneity.network topology | NF-κB | biological oscillations | feedback regulation | paracrine signaling

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASpI294T was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

SummaryNuclear factor kappa B (NF-B) signalling is activated by cellular stress and inflammation and regulates cytokine expression. We applied single-cell imaging to investigate dynamic responses to different doses of tumour necrosis factor alpha (TNF). Lower doses activated fewer cells and those responding showed an increasingly variable delay in the initial NF-B nuclear translocation and associated IB degradation. Robust 100 minute nuclear:cytoplasmic NF-B oscillations were observed over a wide range of TNF concentrations. The result is supported by computational analyses, which identified a limit cycle in the system with a stable 100 minute period over a range of stimuli, and indicated no co-operativity in the pathway activation. These results suggest that a stochastic threshold controls functional all-or-nothing responses in individual cells. Deterministic and stochastic models simulated the experimentally observed activation threshold and gave rise to new predictions about the structure of the system and open the way for better mechanistic understanding of physiological TNF activation of inflammatory responses in cells and tissues.