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.

Nucleocytoplasmic Distribution Is Required for Activation of Resistance by the Potato NB-LRR Receptor Rx1 and Is Balanced by Its Functional Domains

“…Similar diffusional behavior has been found for many chromatinassociated proteins, which led to the idea that most proteins recruited to the chromatin are only binding transiently and show rapid turnover kinetics (Phair et al, 2004;Gorski et al, 2006;Launholt et al, 2006;Hager et al, 2009). A more extensive FRAP analysis than performed here could even provide information about binding kinetics, like the "on" and "off" rates and the relative number of binding sites (Sprague et al, 2004;Sprague and McNally, 2005;Beaudouin et al, 2006). The fact that, like the histone 3 control, the CC fragment Rx1(45-116)-4HA is only released from purified nuclei after sonication in a buffer containing SDS supports the hypothesis that it takes part in an interaction in the nucleus.…”

Early sorting of inner nuclear membrane proteins is conserved

“…Like LBR, nurim is a polytopic protein that accumulates and has limited mobility in the INM (10). Nurim has few amino acids exposed within the cytoplasm or nucleoplasm (12), and there is no evidence that nurim binds to lamins, nuclear pores, or other nucleoplasmic components (10). Mutations made throughout the gene decrease protein accumulation in the INM.…”

“…8 and 11). [4][5][6][7][8][9][10][11][12][13][14][15][16]. When microsomal membranes were prepared from cells infected with recombinant virus-expressing T7-tagged KPNA-4-16, the T7 antibody easily detected KPNA-4-16.…”

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

“…In fluorescence photoactivation, a more recent alternative to FRAP, fluorescence emission is triggered at selected time points by a short illumination pulse. Here, the decrease of fluorescence in the photoactivated region is monitored until the signal reaches equilibrium [4,5].…”