Reduced protein diffusion rate by cytoskeleton in vegetative and polarizedDictyostelium cells

Fluorescence recovery after photobleaching measurements with high spatial r esolution are performed to elucidate the impact of the actin cytoskeleton o n translational mobility of green fluorescent protein (GFP) in aqueous doma ins of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobil ity relative to dilute aqueous solutions. In disrupting the actin filamento us network using latrunculin-A, the intact actin cytoskeletal network is sh own to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindere d protein motions is ascribed to other mechanical barriers and the viscosit y of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore establ ished from measurements under different osmotic conditions. In highly locom otive polarized cells, the obstructing effect of the actin filamentous netw ork is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the p rimary mechanical barrier for protein diffusion and that any noticeable reo rganization of the network is accompanied by altered intracellular protein mobility.