Exclusion of exogenous phosphatidylinositol‐3,4,5‐trisphosphate from neutrophil‐polarizing pseudopodia: stabilization of the uropod and cell polarity

Although there is accumulating evidence that the generation and localization of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have important functions in neutrophil polarization and chemotaxis, the mechanism of this linkage has yet to be established. Here, using exogenous fluorescent PtdIns(3,4,5)P(3) introduced into the inner leaflet of the neutrophil plasma membrane by a cationic carrier, we show that: first, PtdIns(3,4,5)P(3) uniformly delivered to the neutrophil plasma membrane is excluded from newly forming pseudopodia; second, PtdIns(3,4,5)P(3) translocates to and is immobilized at the pole opposite a stable polarizing pseudopod; third, asymmetric delivery of PtdIns(3,4,5)P(3) to the neutrophil triggers the generation of polarizing pseudopodia at the opposite pole; and finally, PtdIns(3,4,5)P(3) triggers repetitive Ca(2+) signals, the onset of which precedes morphological polarization. These data suggest that translocation and immobilization of PtdIns(3,4,5)P(3) or a 3,x-phosphorylated metabolite in the uropod functions as an important polarization cue that defines neutrophil polarity and stabilizes the generation of pseudopodia at the opposite pole

An integrated pharmacological, structural, and genetic analysis of extracellular versus intracellular ROS production in neutrophils

The neutrophil NADPH oxidase produces both intracellular and extracellular reactive oxygen species (ROS). Although oxidase activity is essential for microbial killing, and ROS can act as signaling molecules in the inflammatory process, excessive extracellular ROS directly contributes to inflammatory tissue damage, as well as to cancer progression and immune dysregulation in the tumor microenvironment. How specific signaling pathways contribute to ROS localization is unclear. Here we used a systems pharmacology approach to identify the specific Class I PI3-K isoform p110β, and PLD1, but not PLD2, as critical regulators of extracellular, but not intracellular ROS production in primary neutrophils. Combined crystallographic and molecular dynamics analysis of the PX domain of the oxidase component p47phox, which binds the lipid products of PI 3-K and PLD, was used to clarify the membrane-binding mechanism and guide the design of mutant mice whose p47phox is unable to bind 3-phosphorylated inositol phospholipids. Neutrophils from these K43A mutant animals were specifically deficient in extracellular, but not intracellular, ROS production, and showed increased dependency on signaling through the remaining PLD1 arm. These findings identify the PX domain of p47phox as a critical integrator of PLD1 and p110β signaling for extracellular ROS production, and as a potential therapeutic target for modulating tissue damage and extracellular signaling during inflammation

Genetic Analysis of Sorting Nexins 1 and 2 Reveals a Redundant and Essential Function in Mice

Sorting nexins 1 (Snx1) and 2 (Snx2) are homologues of the yeast gene VPS5 that is required for proper endosome-to-Golgi trafficking. The prevailing thought is that Vps5p is a component of a retrograde trafficking complex called the retromer. Genetic and biochemical evidence suggest mammals may have similar complexes, but their biological role is unknown. Furthermore, if SNX1 and SNX2 belong to such complexes, it is not known whether they act together or separately. Herein, we show that mice lacking SNX1 or SNX2 are viable and fertile, whereas embryos deficient in both proteins arrest at midgestation. These results demonstrate that SNX1 and SNX2 have a highly redundant and necessary function in the mouse. The phenotype of Snx1(-/-);Snx2(-/-) embryos is very similar to that of embryos lacking another retromer homologue, Hβ58. This finding suggests that SNX1/SNX2 and Hβ58 function in the same genetic pathway, providing additional evidence for the existence of mammalian complexes that are structurally similar to the yeast retromer. Furthermore, the viability of Snx1(-/-) and Snx2(-/-) mice demonstrates that it is not necessary for SNX1 and SNX2 to act together. Electron microscopy indicates morphological alterations of apical intracellular compartments in the Snx1(-/-);Snx2(-/-) yolk-sac visceral endoderm, suggesting SNX1 and SNX2 may be required for proper cellular trafficking. However, tetraploid aggregation experiments suggest that yolk sac defects cannot fully account for Snx1(-/-); Snx2(-/-) embryonic lethality. Furthermore, endocytosis of transferrin and low-density lipoprotein is unaffected in mutant primary embryonic fibroblasts, indicating that SNX1 and SNX2 are not essential for endocytosis in all cells. Although the two proteins demonstrate functional redundancy, Snx1(+/-);Snx2(-/-) mice display abnormalities not observed in Snx1(-/-);Snx2(+/-) mice, revealing that SNX1 and SNX2, or their genetic regulation, are not equivalent. Significantly, these studies represent the first mutations in the mammalian sorting nexin gene family and indicate that sorting nexins perform essential functions in mammals