The first morphological change after neuronal differentiation is the microtubule-dependent initiation of thin cell protrusions called neurites. Here we performed a siRNA-based morphometric screen in P19 stem cells to evaluate the role of 408 microtubule-regulating genes during this early neuromorphogenesis step. This screen uncovered several novel regulatory factors, including specific complex subunits of the microtubule motor dynein involved in neurite initiation and a novel role for the microtubule end-binding protein EB2 in attenuation of neurite outgrowth. Epistasis analysis suggests that competition between EB1 and EB2 regulates neurite length, which links its expression to neurite outgrowth. We propose a model that explains how microtubule regulators can mediate cellular morphogenesis during the early steps of neuronal development by controlling microtubule stabilization and organizing dynein-generated forces.
Abstract Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by a decrease in migration distance and an increase in migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.
Abstract Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.
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