An Order of Magnitude Faster AIP1-Associated Actin Disruption than Nucleation by the Arp2/3 Complex in Lamellipodia

The mechanism of lamellipod actin turnover is still under debate. To clarify the intracellular behavior of the recently-identified actin disruption mechanism, we examined kinetics of AIP1 using fluorescent single-molecule speckle microscopy. AIP1 is thought to cap cofilin-generated actin barbed ends. Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase. Moreover, actin-associated AIP1 was rapidly abolished by jasplakinolide, which concurrently blocked the F-actin-cofilin interaction. Jasplakinolide also slowed dissociation of AIP1, which is analogous to the effect of this drug on capping protein. These findings provide in vivo evidence of the association of AIP1 with barbed ends generated by cofilin-catalyzed filament disruption. Single-molecule observation found distribution of F-actin-associated AIP1 throughout lamellipodia, and revealed even faster dissociation of AIP1 than capping protein. The estimated overall AIP1-associated actin disruption rate, 1.8 µM/s, was one order of magnitude faster than Arp2/3 complex-catalyzed actin nucleation in lamellipodia. This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis. Our data together with recent biochemical studies imply barbed end-preferred frequent filament disruption. Frequent generation of AIP1-associated barbed ends and subsequent release of AIP1 may be the mechanism that facilitates previously observed ubiquitous actin polymerization throughout lamellipodia

Cdc42 and Rac Family GTPases Regulate Mode and Speed but Not Direction of Primary Fibroblast Migration during Platelet-Derived Growth Factor-Dependent Chemotaxis ▿ †

Cdc42 and Rac family GTPases are important regulators of morphology, motility, and polarity in a variety of mammalian cell types. However, comprehensive analysis of their roles in the morphological and behavioral aspects of chemotaxis within a single experimental system is still lacking. Here we demonstrate using a direct viewing chemotaxis assay that of all of the Cdc42/Rac1-related GTPases expressed in primary fibroblasts, Cdc42, Rac1, and RhoG are required for efficient migration towards platelet-derived growth factor (PDGF). During migration, Cdc42-, Rac1-, and RhoG-deficient cells show aberrant morphology characterized as cell elongation and cell body rounding, loss of lamellipodia, and formation of thick membrane extensions, respectively. Analysis of individual cell trajectories reveals that cell speed is significantly reduced, as well as persistence, but to a smaller degree, while the directional response to the gradient of PDGF is not affected. Combined knockdown of Cdc42, Rac1, and RhoG results in greater inhibition of cell speed than when each protein is knocked down alone, but the cells are still capable of migrating toward PDGF. We conclude that, Cdc42, Rac1, and RhoG function cooperatively during cell migration and that, while each GTPase is implicated in the control of morphology and cell speed, these and other Cdc42/Rac-related GTPases are not essential for the directional response toward PDGF

Flanker task paradigm.

<p>Each trial involved both eyes and two dots. The trial began with a fixation cross. After a random duration of 400 or 600 ms, lateral eye movements were presented as flanker distractors. Then, a color alteration of the dot followed after a random duration of 400 or 600 ms. The target stimulus was a red dot, which vanished immediately after the participant’s response (i.e., button pressing). Finally, a fixation cross was presented again for 400 or 600 ms before the next trial. A total of 148 trials (74 congruent trials and 74 incongruent trials) were presented in a randomized order. The total time of the task was approximately 7 min.</p

Conflict duration on the flanker task (i.e., reaction time for each incongruent trial–reaction time for each congruent trial) pre- (white bar) and post-treatment (black bar) with OT or placebo.

<p>Two-way ANOVA (treatment × drug) failed to demonstrate any drug or treatment effects, and no significant interaction between these two factors was detected. Therefore, we failed to demonstrate any OT-related effect on attentional-inhibitory control. The error bars represent 1 standard error.</p

The hostility detection ratio (a lower value indicates a positively valenced interpretation of the facial expression) pre- (white bar) and post-treatment (black bar) with OT or placebo.

<p>A, happy face condition. B, angry face condition. C, ambiguous face condition. D, neutral face condition. Two-way ANOVA (treatment × drug) revealed significant treatment effects (i.e., order effects) under the ambiguous and neutral conditions but not under the happy or angry condition. We failed to detect an OT-related effect on the hostility detection ratio under any condition. The error bars represent 1 standard error.</p

Scatter plot of the OT-induced changes on the facial emotion recognition task and on the flanker task.

<p>The vertical axis represents the OT-induced changes (i.e., OT [post—pre]—placebo [post—pre]) in the interpretation of facial expression for the combined uncertain (i.e., neutral and ambiguous) conditions. The horizontal axis represents the OT-induced changes (i.e., OT [post—pre]—placebo [post—pre]) in conflict duration on the flanker task. Notably, the change to less-hostile facial cognition was associated with enhanced attentional-inhibitory control after OT administration. The solid line denotes the regression line.</p