Mapping eGFP oligomer mobility in living cell nuclei

Area of Science:

• Cell Biology
• Biophysics
• Molecular Dynamics

Background:

• Particle mobility in cell nuclei is influenced by nuclear viscosity, flows, active transport, and obstacles like the chromatin network.
• Understanding diffusion dynamics is crucial for comprehending nuclear processes.

Purpose of the Study:

• To investigate if chromatin density affects the mobility of small fluorescent proteins within living cell nuclei.
• To establish reliable methods for studying diffusion in live cells using fluorescence correlation spectroscopy (FCS).

Main Methods:

• Utilized two-color confocal scanning fluorescence correlation spectroscopy (FCS) to study inert fluorescent protein diffusion in living cell nuclei.
• Developed strategies to mitigate FCS-specific artifacts in live-cell studies, including fluorophore calibration, temperature, and acquisition settings.
• Employed a series of green fluorescent protein (GFP) oligomers (monomer to tetramer) as inert tracers.

Main Results:

• Identified and addressed FCS artifacts crucial for accurate live-cell measurements.
• Observed significant variations in GFP mobility within the nucleus across different human cell lines, but found no correlation with chromatin density.
• Demonstrated that intranuclear diffusion is highly dependent on protein size, with diffusion coefficients decreasing significantly for GFP oligomers compared to free diffusion.
• Confirmed that the entire chromatin network is accessible to small proteins up to the size of eGFP-tetramers, irrespective of chromatin density or cell type.

Conclusions:

• Chromatin density does not impede the diffusion of small fluorescent proteins within the cell nucleus.
• Small proteins, including GFP monomers and multimers, can freely access even the densest chromatin regions.
• Protein size is a critical factor influencing intranuclear mobility, more so than previously expected for free diffusion.