Dissecting the Contribution of Diffusion and Interactions to the Mobility of Nuclear Proteins

Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from su...

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Bibliographic Details
Published in:Biophysical journal 2006-03, Vol.90 (6), p.1878-1894
Main Authors: Beaudouin, Joël, Mora-Bermúdez, Felipe, Klee, Thorsten, Daigle, Nathalie, Ellenberg, Jan
Format: Article
Language:English
Subjects:
Active Transport, Cell Nucleus - physiology
Animals
Biophysical Theory and Modeling
Biophysics
Cells, Cultured
Chromatin
Computer Simulation
Diffusion
Kidney - chemistry
Kidney - metabolism
Microscopy, Fluorescence - methods
Models, Biological
Models, Chemical
Motion
Nuclear Proteins - chemistry
Nuclear Proteins - metabolism
Protein Interaction Mapping - methods
Proteins
Rats
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Summary:Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells.
ISSN:0006-3495
1542-0086
DOI:10.1529/biophysj.105.071241