Reduced protein diffusion rate by cytoskeleton in vegetative and polarized Dictyostelium cells

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

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and-unexpectedly-lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractant