Comparing the intracellular mobility of fluorescent proteins following in vitro expression or cell loading with...

The application of fluorescent proteins in live cells has greatly improved our ability to study molecular mobility, which both reflects molecular function in live cells and reveals the properties of the local environment. Although measuring molecular mobility with fluorescent fusion proteins is powerful and convenient, certain experiments still require exogenous macromolecules to be loaded into cells. Cell viability provides a rough gauge of cellular damage following membrane permeabilization, but it is unknown how permeabilization will affect intracellular mobility. We have used fluorescence correlation spectroscopy to measure the intracellular dynamics of the enhanced green fluorescent protein (EGFP) in living human embryonic kidney (HEK) cells under conditions where the EGFP is either expressed or loaded using streptolysin O (SLO) permeabilization to determine how permeabilization effects mobility. We found that purified EGFP loaded with SLO has the same mobility as the expressed EGFP, while the mobility of the expressed EGFP after SLO permeabilization treatment becomes slightly slower. Our results indicate that SLO permeabilization is often accompanied by the loss of cellular soluble proteins to the surrounding medium, which explains the apparent decrease in diffusion rates following treatment. These measurements are also relevant to the role of molecular crowding in the intracellular mobility of proteins.