Evaluating Intracellular Crowded with a Glycine-Inserted Mutant Fluorescent Protein

Abstract

The cell environment is very crowded, containing various molecules, proteins and nucleotides. This crowded condition is an indispensable factor for cellular functions of proteins. In the past, the diffusion coefficient of a chemical probe has been used as an evaluation index of the intracellular crowded condition. However, crowding depends not only on the mobility, but also the density of the crowding agents. We have succeeded in making a yellow fluorescent protein (YFP) that senses crowding density via hydrophobicity by inserting into the YFP a glycine and conjugating it to cyan fluorescent protein (CFP), which is insensitive to Forster resonance energy transfer (FRET) probe. This probe has been named GimRET (Glycine inserted mutant FRET probe). GimRET enabled us to visualize the dynamic changes of the intracellular crowding density during cell division. Because GimRET can distinguish the crowding density from the viscosity of the solution, crowding can be evaluated by the GimRET fluorescence intensity ratio and diffusion coefficient, which respectively reflects the density and mobility of the crowding agents. While the diffusion coefficient of GimRET linearly relates to the fluorescence intensity ratio, the slope also depends on the location of the cell, i.e., nucleus or cytoplasm, indicating that the diffusion coefficient alone is insufficient for defining crowding. Here, we propose the simultaneous observation of the GimRET intensity ratio and the diffusion coefficient as a way to evaluate intracellular crowding.