Quantitative evaluation of macromolecular crowding environment based on translational and rotational diffusion using...

Area of Science:

• Biophysics
• Cell Biology
• Physical Chemistry

Background:

• Macromolecular crowding (MMC) significantly impacts cellular processes.
• Accurate characterization of the cellular nano-environment is crucial for understanding MMC.
• Existing methods for evaluating MMC nano-environments have limitations.

Purpose of the Study:

• To introduce and validate polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) as a method for evaluating MMC nano-environments.
• To compare the nano-environments of various solutions (small molecules, gels, MMC solutions) using Pol-FCS.
• To assess the applicability of Pol-FCS in living cells and other nano-environments like liquid-liquid phase separation.

Main Methods:

• Utilizing Pol-FCS to simultaneously measure rotational and translational diffusion of fluorescent molecules.
• Applying Pol-FCS to analyze solutions of small molecules, gels, and MMC solutions.
• Investigating the nano-environment within the cytosol and nucleus of living cells across different cell lines and cell cycles.

Main Results:

• Pol-FCS successfully differentiated the nano-environments of small molecules, gels, and MMC solutions.
• The method detected phase shifts in polyethylene glycol solutions.
• Distinct nano-environmental characteristics were observed in the cytosol and nucleus of living cells.

Conclusions:

• Pol-FCS is a valuable tool for characterizing nano-environments in MMC solutions.
• The technique offers a low-damage approach for in vivo analysis of cellular nano-environments.
• Pol-FCS shows potential for studying other complex nano-environments, including liquid-liquid phase separation.