Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins.

Abstract:

: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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Beaudouin J,Mora-Bermúdez F,Klee T,Daigle N,Ellenberg J

doi

10.1529/biophysj.105.071241

subject

Has Abstract

pub_date

2006-03-15 00:00:00

pages

1878-94

issue

6

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(06)72375-X

journal_volume

90

pub_type

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