Sources of Cell-to-cell Variability in Canonical Nuclear Factor-κB (NF-κB) Signaling Pathway Inferred from Single Cell Dynamic Images

Abstract

The canonical nuclear factor-B (NF-B) signaling pathway controls a gene network important in the cellular inflammatory response. Upon activation, NF-B/RelA is released from cytoplasmic inhibitors, from where it translocates into the nucleus, subsequently activating negative feedback loops producing either monophasic or damped oscillatory nucleo-cytoplasmic dynamics. Although the population behavior of the NF-B pathway has been extensively modeled, the sources of cell-to-cell variability are not well understood. We describe an integrated experimental-computational analysis of NF-B/RelA translocation in a validated cell model exhibiting monophasic dynamics. Quantitative measures of cellular geometry and total cytoplasmic concentration and translocated RelA amounts were used as priors in Bayesian inference to estimate biophysically realistic parameter values based on dynamic live cell imaging studies of enhanced GFP-tagged RelA in stable transfectants. Bayesian inference was performed on multiple cells simultaneously, assuming identical reaction rate parameters, whereas cellular geometry and initial and total NF-B concentration-related parameters were cell-specific. A subpopulation of cells exhibiting distinct kinetic profiles was identified that corresponded to differences in the IB␣ translation rate. We conclude that cellular geometry, initial and total NF-B concentration, IB␣ translation, and IB␣ degradation rates account for distinct cell-to-cell differences in canonical NF-B translocation dynamics.

Inducible Tumor Necrosis Factor (TNF) Receptor-associated Factor-1 Expression Couples the Canonical to the Non-canonical NF-κB Pathway in TNF Stimulation

“…1A). Live cell imaging data at a single cell resolution clearly demonstrate a robust increase in the nuclear RelA translocation after TNF stimulation measured by confocal microscopy (24). By contrast, p52 translocation was significantly increased after 3 h of stimulation (Fig.…”

BRD4 Couples NF-κB/RelA with Airway Inflammation and the IRF-RIG-I Amplification Loop in Respiratory Syncytial Virus Infection

“…hSAECs were plated on cover glasses pretreated with rat tail collagen (Roche Applied Sciences). After indicated infection times and treatments, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized by 0.1% Triton X-100, blocked, and incubated with indicated primary rabbit polyclonal Ab as described previously (18,78). After incubation with Alexa goat anti-rabbit Ab, cells were washed and counterstained with 4=,6-diamidino-2-phenylindole (DAPI).…”

RelA Ser276 Phosphorylation-Coupled Lys310 Acetylation Controls Transcriptional Elongation of Inflammatory Cytokines in Respiratory Syncytial Virus Infection

“…Retroviral RelA expression. Full-length human RelA and site mutants were expressed as FLAG-enhanced green fluorescent protein (EGFP) fusion proteins using the pCX4-Pur expression vector (2,27). Retrovirus stocks produced in Bosc23 cells were cotransfected with pCX4-pur-EGFP-RelA and amphotrophic packaging plasmid pCL-10A1 (37).…”