G protein-coupled receptor-mediated membrane targeting of PLCγ2 is essential for neutrophil chemotaxis.

The current dogma is that chemoattractants G protein-coupled receptors activate ß phospholipase C while receptor tyrosine kinases activate γ phospholipase C. Here, we show that chemoattractant/G protein-coupled receptor-mediated membrane recruitment of γ2 phospholipase C constitutes G protein-coupled receptor-mediated phospholipase C signaling and is essential for neutrophil polarization and migration during chemotaxis. In response to a chemoattractant stimulation, cells lacking γ2 phospholipase C (plcg2kd) displayed altered dynamics of diacylglycerol production and calcium response, increased Ras/PI3K/Akt activation, elevated GSK3 phosphorylation and cofilin activation, impaired dynamics of actin polymerization, and, consequently, defects in cell polarization and migration during chemotaxis. The study reveals a molecular mechanism of membrane targeting of γ2 phospholipase C and the signaling pathways by which γ2 phospholipase C plays an essential role in neutrophil chemotaxis.

Ras inhibitor CAPRI enables neutrophil-like cells to chemotax through a higher-concentration range of gradients.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.

Membrane Targeting of C2GAP1 Enables Dictyostelium discoideum to Sense Chemoattractant Gradient at a Higher Concentration Range.

Chemotaxis, which is G protein-coupled receptor (GPCR)-mediated directional cell migration, plays pivotal roles in diverse human diseases, including recruitment of leukocytes to inflammation sites and metastasis of cancer. It is still not fully understood how eukaryotes sense and chemotax in response to chemoattractants with an enormous concentration range. A genetically traceable model organism, Dictyostelium discoideum, is the best-studied organism for GPCR-mediated chemotaxis. Recently, we have shown that C2GAP1 controls G protein coupled receptor-mediated Ras adaptation and chemotaxis. Here, we investigated the molecular mechanism and the biological function of C2GAP1 membrane targeting for chemotaxis. We show that calcium and phospholipids on the plasma membrane play critical roles in membrane targeting of C2GAP1. Cells lacking C2GAP1 (c2gapA -) displayed an improved chemotaxis in response to chemoattractant gradients at subsensitive or low concentrations (<100 nM), while exhibiting impaired chemotaxis in response to gradients at high concentrations (>1 µM). Taken together, our results demonstrate that the membrane targeting of C2GAP1 enables Dictyostelium to sense chemoattractant gradients at a higher concentration range. This mechanism is likely an evolutionarily conserved molecular mechanism of Ras regulation in the adaptation and chemotaxis of eukaryotes.

Visualizing Key Signaling Components of Macropinocytosis and Phagocytosis Using Confocal Microscopy in the Model Organism Dictyostelium discoideum.

Macropinocytosis and phagocytosis are the processes by which eukaryotic cells use their plasma membrane to engulf liquid or a large particle and give rise to an internal compartment called the macropinosomes or phagosome, respectively. Dictyostelium discoideum provides a powerful system to understand the molecular mechanism of these two fundamental cellular processes that impact human health and disease. Recent developments in fluorescence microscopy allow direct visualization of intracellular signaling events with high temporal and spatial resolution. Here, we describe methods to visualize temporospatial activation or localization of key signaling components that are crucial for macropinocytosis and phagocytosis using confocal fluorescence microscopy.