Reduced protein diffusion rate as revealed with fluorescence recovery after photobleaching of the green fluorescent protein in live cells

Fluorescence recovery after photobleaching measurements are performed to elucidate the mobility of the green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. A confocal detection scheme is adopted that allows one to record the recovery dynamics which originate exclusively from intracellular regions. In the confocal arrangement, favorable excitation intensity levels can be applied that limit photoinduced cellular damage. Results are analyzed with a versatile model that does not rely on the commonly applied Gaussian approximation of the illumination volume. In vegetative Dictyostelium cells, GFP molecules experience a 3.6 fold reduction of their translational mobility relative to the diffusion in dilute aqueous solutions. In depolymerizing the actin filaments using Latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.4 cP, which accounts for 53% of the restrained molecular diffusion of GFP. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells the obstructing effect of the actin filamentous network is seen to decline to 0.5 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied with an altered intracellular protein mobility. Parts of this chapter can also be found in the following paper: E. O. Potma, L. Bosgraaf, J. Roelofs, W. P. de Boeij, P. J. M. van Haastert and D. A. Wiersma, “Reduced protein diffusion rate by cytoskeleton in vegetative and polarized Dictyostelium cells”, Biophys. J. 81, in press.