Abstract
Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation. Modulating TNFα concentration or pulse duration yielded complex, reproducible, and differential ΙκBα dynamics in both cell populations and live single cells. Tremendous heterogeneity in the transcriptional amplitudes of individual responding cells was observed, which was greater than the heterogeneity in the transcriptional kinetics of responsive cells. Furthermore, administration of various TNFα doses in vivo generated ΙκBα dynamic profiles in the liver resembling those observed in single cells and populations of cells stimulated with TNFα pulses. This suggested that dose modulation of circulating TNFα was perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a robust bioluminescent reporter strategy enabled rigorous quantitation of NF-κB/ΙκBα dynamics in both live single cells and cell populations and furthermore, revealed reproducible behaviors that informed interpretation of in vivo studies.
Highlights
Understanding the biological significance of feedback loops requires interrogation at multiple scales
Using a B53 IB␣-firefly luciferase (FLuc) bioluminescent reporter, we rigorously evaluated the dynamics of ⌱B␣ degradation and subsequent nuclear factor B (NF-B) transcriptional activity in response to diverse modes of TNF␣ stimulation
Circulating tumor necrosis factor ␣ (TNF␣) doses are perceived by the liver as pulses
Summary
Circulating tumor necrosis factor ␣ (TNF␣) doses are perceived by the liver as pulses. We have developed a dynamic bioluminescent reporter strategy that enables correlative quantitation of the NF-B/⌱B␣ negative feedback loop in single cells, cell populations, and at the tissue level in live animals. The synthetic B5 promoter has enhanced sensitivity that enables measurement of subtle changes in transcriptional dynamics. This reporter strategy provides a real time dynamic link between fluorescence-based reporters that measure NF-B nuclear shuttling in single cells and conventional destructive techniques (quantitative-PCR of target genes, transcriptional profiling, EMSA, etc.) that measure downstream NF-B transcriptional activity in cell populations. We have exploited the unique characteristics of the B53⌱B␣-FLuc reporter for multiscale interrogation of the negative feedback loop in single cells, cell populations, and in vivo. Dynamics in both single cells and populations of cells, and we discovered reproducible behaviors that informed interpretation of in vivo studies
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
