Macromolecular crowding affects protein diffusion in stress fibers as revealed by fluorescence correlation spectroscopy

Abstract The diffusion of cell components such as proteins is crucial to the function of all living cells. The abundance of macromolecules in the cell is likely to cause a state of macromolecular crowding, but its effects on the extent of diffusion remain poorly understood. Here we investigate the diffusion rate in three distinct locations in mesenchymal cell types, namely the cytoplasm, the stress fibers, and those below the nucleus using three kinds of inert green fluorescent proteins (GFPs), namely a monomer, dimer, and trimer GFP. Fluorescence correlation spectroscopy (FCS) was used to determine the diffusion coefficients. We show that diffusion tends to be lowered on average in stress fibers and is significantly lower in those located below the nucleus. Our data suggests that the diffusive properties of GFPs, and potentially other molecules as well, are hindered by macromolecular crowding. However, although the size dependence on protein diffusion was also studied for monomer, dimer, and trimer GFPs, there was no significant difference in the diffusion rates among the GFPs of these sizes. These results could be attributed to the lack of significant change in protein size among the selected GFP multimers. Nevertheless, the data presented here would provide a basis for better understanding of complicated protein diffusion in the nonuniform cytoplasm and in turn of cellular responses to mechanical stress as well as their local mechanical properties.