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

¹ School of Biological Sciences, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK
² AstraZeneca R&D Charnwood, Molecular Biology, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK
³ Kinetic Imaging Ltd, 2 Brunel Road, Wirral, CH62 3NY, UK

^* Author for correspondence (e-mail: mwhite{at}liv.ac.uk )

Accepted 13 December 2001

Proteins of the NF-B transcription factor family normally reside in the cytoplasm of cells in a complex with IB inhibitor proteins. Stimulation with TNF leads to proteosomal degradation of the IB proteins and nuclear translocation of the NF-B proteins. Expression of p65 and IB fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IB-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression, the half-time of IB-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 IB and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IB is the true substrate for TNF stimulation in mammalian cells.

When cells were treated with the CRM-1-dependent nuclear export inhibitor, leptomycin B (LMB), there was nuclear accumulation of IB-EGFP and p65-dsRed, with IB-EGFP accumulating more rapidly. No NF-B-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IB-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNF, degradation of IB-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-B-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-B-dependent transcription occur even when nuclear export of NF-B 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.

Key words: NF-B, Signal transduction, Fluorescent protein fusions, Firefly luciferase, Luminescence imaging, Confocal microscopy

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