This study investigates the polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Using a fluorescently tagged version of the PH domain of AKT (PHAKT-GFP), researchers observed that this protein is recruited to the leading edge of neutrophils in response to chemoattractants, indicating an internal signaling gradient steeper than the external chemoattractant gradient. The translocation of PHAKT-GFP was inhibited by a toxin from Clostridium difficile, suggesting it requires the activity of Rho GTPases. Neutrophils and other motile cells respond to chemoattractant gradients by rapidly adopting a polarized morphology, with distinct leading and trailing edges. Actin is polymerized preferentially at the leading edge, even in shallow gradients. The asymmetry of newly polymerized actin suggests that neutrophils can greatly amplify the smaller asymmetry of the extracellular signal detected by chemoattractant receptors. This amplification must occur between receptor activation and actin polymerization, as receptors remain uniformly distributed during chemotaxis. The study used a fluorescent probe to examine the spatial distribution of an intermediate intracellular signal and found that this mechanism depends on the activity of one or more Rho GTPases and likely requires activation of phosphatidylinositol 3-kinase (PI3K). In a soil amoeba, Dictyostelium discoideum, asymmetric recruitment of two signal transduction proteins to the cell surface occurs without actin polymerization. However, in neutrophils, the recruitment of PHAKT-GFP to the plasma membrane is asymmetric and depends on Rho GTPases. In HL-60 cells, PHAKT-GFP translocated to the plasma membrane upon exposure to chemoattractants, and this translocation was rapid and transient. In a gradient of fMLP, PHAKT-GFP was recruited exclusively to the parts of the cell surface that received the strongest stimulation, tightly accompanying actin polymerization and pseudopod formation. The internal gradient of PHAKT-GFP distribution was steeper than that of the extracellular stimulus. Exposure to latrunculin-B, a toxin that sequesters monomeric actin, caused depolymerization of the actin cytoskeleton, but PHAKT-GFP still translocated asymmetrically to the face closest to the chemoattractant source. This suggests that the signaling machinery of neutrophils can amplify the external signaling gradient independently of actin polymerization. The study also found that Rho GTPases are required for the recruitment of PHAKT-GFP to the plasma membrane. A toxin from Clostridium difficile inactivates Rho GTPases and inhibits PHAKT-GFP translocation and membrane ruffling. However, in the absence of toxin treatment, a uniform concentration of chemoatThis study investigates the polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Using a fluorescently tagged version of the PH domain of AKT (PHAKT-GFP), researchers observed that this protein is recruited to the leading edge of neutrophils in response to chemoattractants, indicating an internal signaling gradient steeper than the external chemoattractant gradient. The translocation of PHAKT-GFP was inhibited by a toxin from Clostridium difficile, suggesting it requires the activity of Rho GTPases. Neutrophils and other motile cells respond to chemoattractant gradients by rapidly adopting a polarized morphology, with distinct leading and trailing edges. Actin is polymerized preferentially at the leading edge, even in shallow gradients. The asymmetry of newly polymerized actin suggests that neutrophils can greatly amplify the smaller asymmetry of the extracellular signal detected by chemoattractant receptors. This amplification must occur between receptor activation and actin polymerization, as receptors remain uniformly distributed during chemotaxis. The study used a fluorescent probe to examine the spatial distribution of an intermediate intracellular signal and found that this mechanism depends on the activity of one or more Rho GTPases and likely requires activation of phosphatidylinositol 3-kinase (PI3K). In a soil amoeba, Dictyostelium discoideum, asymmetric recruitment of two signal transduction proteins to the cell surface occurs without actin polymerization. However, in neutrophils, the recruitment of PHAKT-GFP to the plasma membrane is asymmetric and depends on Rho GTPases. In HL-60 cells, PHAKT-GFP translocated to the plasma membrane upon exposure to chemoattractants, and this translocation was rapid and transient. In a gradient of fMLP, PHAKT-GFP was recruited exclusively to the parts of the cell surface that received the strongest stimulation, tightly accompanying actin polymerization and pseudopod formation. The internal gradient of PHAKT-GFP distribution was steeper than that of the extracellular stimulus. Exposure to latrunculin-B, a toxin that sequesters monomeric actin, caused depolymerization of the actin cytoskeleton, but PHAKT-GFP still translocated asymmetrically to the face closest to the chemoattractant source. This suggests that the signaling machinery of neutrophils can amplify the external signaling gradient independently of actin polymerization. The study also found that Rho GTPases are required for the recruitment of PHAKT-GFP to the plasma membrane. A toxin from Clostridium difficile inactivates Rho GTPases and inhibits PHAKT-GFP translocation and membrane ruffling. However, in the absence of toxin treatment, a uniform concentration of chemoat