Abstract
Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers.
Highlights
The accessibility to compartments and the global binding energy landscape faced by biomolecules are important parameters determining their function, and their quantification is an essential task for cell biology
Since the diffusion of small proteins in the cell nucleus is central to their mechanism of action as well as to understanding nuclear architecture, we studied how diffusion of such macromolecules is affected by the chromatin network
The fluorescence observed in the cells originates only from the complete eGFP-constructs, as previously stated in [30] and shown here with the help of native gels and immunoblotting
Summary
The accessibility to compartments and the global binding energy landscape faced by biomolecules are important parameters determining their function, and their quantification is an essential task for cell biology. Recent studies demonstrate that proteins show anomalous diffusion – i.e. a mean-square displacement whose time dependence is weaker than linear – in the cytoplasm [2] as well as in the nucleus of living cells [3]. This implies either geometrically obstructed or spatially confined motion [4,5,6]. The diffusion in the nuclei of living cells is affected by the distribution and the density of the intranuclear obstacles, the transient binding of the proteins to these obstacles, the local viscosity or active transport phenomena. Since the diffusion of small proteins in the cell nucleus is central to their mechanism of action as well as to understanding nuclear architecture, we studied how diffusion of such macromolecules is affected by the chromatin network
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