Nearly identical cells can exhibit substantially different responses to the same stimulus. We monitored the nuclear localization dynamics of nuclear factor κB (NF-κB) in single cells stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS). Cells stimulated with TNF-α have quantitative differences in NF-κB nuclear localization, whereas LPS-stimulated cells can be clustered into transient or persistent responders, representing two qualitatively different groups based on the NF-κB response. These distinct behaviors can be linked to a secondary paracrine signal secreted at low concentrations, such that not all cells undergo a second round of NF-κB activation. From our single-cell data, we built a computational model that captures cell variability, as well as population behaviors. Our findings demonstrate that mammalian cells can create “noisy” environments in order to produce diversified responses to stimuli.

NF-kB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF-kB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF-kB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane-associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD-deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF-kB activation by aCD3/aCD28 cross-linking and PMA/ionomycin treatment, but not by TNFa or dsRNA. CARD11 is not required for TCR-mediated induction of NFAT or AP-1. CARD11 functions upstream of the IkB-kinase (IKK) complex and cooperates with Bcl10 in a CARD domain-dependent manner. RNAi-rescue experiments suggest that the CARD, coiled-coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factorspeci®c activation of NF-kB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.