Multi-parameter analysis of the kinetics of NF-kappaB signalling and transcription in single living cells.

Proteins of the NF-kappaB transcription factor family normally reside in the cytoplasm of cells in a complex with IkappaB inhibitor proteins. Stimulation with TNFalpha leads to proteosomal degradation of the IkappaB proteins and nuclear translocation of the NF-kappaB proteins. Expression of p65 and IkappaBalpha fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IkappaBalpha-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression, the half-time of IkappaBalpha-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IkappaBalpha and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IkappaBalpha is the true substrate for TNFalpha stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor, leptomycin B (LMB), there was nuclear accumulation of IkappaBalpha-EGFP and p65-dsRed, with IkappaBalpha-EGFP accumulating more rapidly. No NF-kappaB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IkappaBalpha-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFalpha, degradation of IkappaBalpha-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-kappaB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-kappaB-dependent transcription occur even when nuclear export of NF-kappaB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.