Reduced Protein Diffusion Rate by Cytoskeleton in Vegetative and Polarized Dictyostelium Cells

Abstract

Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. 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 0.46 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 by altered intracellular protein mobility.

Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

“…The algorithm provides as an output the distribution of the obtained apparent diffusion constants for a specific condition. To test the robustness of the algorithm, we used free eGFP diffusing in aqueous solution, for which we obtained a single peak distribution centred around 92 μm 2 /s, a value which is in agreement with previous studies (Potma et al , 2001). …”

“…As expected, the average autocorrelation curve fits well with a one‐component free diffusion model (Fig 4A). The single peak of the frequency distribution of diffusion constants, generated by the Maximum Entropy approach (Materials and Methods), was centred around 92 μm 2 /s (Fig 4C), as previously described (Potma et al , 2001). Next, we analysed transiently expressed eGFP in living cells, whose average autocorrelation curve also fitted a one‐component free diffusion model (Fig 4B), as expected for a protein with no specific interactions with the nuclear environment.…”

Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium

“…2). Although the axonemal components will reduce the effective cross-sectional area available for diffusion, as the maximal diameter of each microtubule The aqueous diffusion coefficient of EGFP, D aq, EGFP , was reported previously to be 87 (Swaminathan et al, 1997;Brown et al, 1999;Potma et al, 2001) and 91 µm 2 s 1 (Calvert et al, 2007). D aq, PAGFP was reported to be 89 µm 2 s 1 (Calvert et al, 2007).…”

New insights into diffusion in 3D crowded media by Monte Carlo simulations: effect of size, mobility and spatial distribution of obstacles

“…A great deal of information about motion of molecules in living cells has been obtained from intracellular measurements using different experimental techniques 13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and from simulations 14,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] . Experimental data are usually obtained by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) techniques.…”