Lunhua Liu

mTORC2 Regulates Neutrophil Chemotaxis in a cAMP- and RhoA-Dependent Fashion

We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA-signaling axis, independently of actin reorganization during neutrophil chemotaxis.

A Gβγ Effector, ElmoE, Transduces GPCR Signaling to the Actin Network during Chemotaxis

Activation of G protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gβγ subunits regulate actin assembly and migration, but the protein(s) linking Gβγ to the actin cytoskeleton remains unknown. Here, we identified a Gβγ effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE associates with Gβγ and Dock-like proteins to activate the small GTPase Rac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin. Thus, ElmoE serves as a link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gβγ, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis.

Glia maturation factor-γ mediates neutrophil chemotaxis

Chemotaxis is fundamental to the directional migration of neutrophils toward endogenous and exogenous chemoattractants. Recent studies have demonstrated that ADF/cofilin superfamily members play important roles in reorganizing the actin cytoskeleton by disassembling actin filaments. GMFG, a novel ADF/cofilin superfamily protein that is expressed in inflammatory cells, has been implicated in regulating actin reorganization in microendothelial cells, but its function in neutrophils remains unclear. Here, we show that GMFG is an important regulator for cell migration and polarity in neutrophils. Knockdown of endogenous GMFG impaired fMLF‐ and IL‐8 (CXCL8)‐induced chemotaxis in dHL‐60 cells. GMFG knockdown attenuated the formation of lamellipodia at the leading edge of cells exposed to fMLF or CXCL8, as well as the phosphorylation of p38 and PAK1/2 in response to fMLF or CXCL8. Live cell imaging revealed that GMFG was recruited to the leading edge of cells in response to fMLF, as well as CXCL8. Overexpression of GMFG enhanced phosphorylation of p38 but not of PAK1/2 in dHL‐60 cells. In addition, we found that GMFG is associated with WAVE2. Taken together, our findings suggest that GMFG is a novel factor in regulating neutrophil chemotaxis by modulating actin cytoskeleton reorganization.

PKCβII acts downstream of chemoattractant receptors and mTORC2 to regulate cAMP production and myosin II activity in neutrophils

mTORC2 has been shown to be involved in cytoskeletal regulation, but the mechanisms by which this takes place are poorly understood. This study shows that PKCβII is specifically required for mTORC2-dependent activation of adenylyl cyclase 9 and back retraction during neutrophil chemotaxis to chemoattractants.

Radil controls neutrophil adhesion and motility through β2-integrin activation.

Various agonists trigger β2-integrin activation in neutrophils, yet the mechanisms that regulate β2-integrin inside-out signaling remain obscure. Radil, a novel Rap downstream effector, is an important adapter in the pathway that links G protein–coupled chemoattractant receptors to adhesion complexes during neutrophil chemotaxis.

Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1

Background Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-activating and chemoattractant chemokine. We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure. Methods Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control. Results Redistribution of ZO-1 and an influx of CD11b+Lys6G+ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP+ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe. We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration. Conclusions Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process.

A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration

Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.

Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin

Monocyte migration requires the dynamic redistribution of integrins through a regulated endo-exocytosis cycle, but the complex molecular mechanisms underlying this process have not been fully elucidated. Glia maturation factor-γ (GMFG), a novel regulator of the Arp2/3 complex, has been shown to regulate directional migration of neutrophils and T-lymphocytes. In this study, we explored the important role of GMFG in monocyte chemotaxis, adhesion, and β1-integrin turnover. We found that knockdown of GMFG in monocytes resulted in impaired chemotactic migration toward formyl-Met-Leu-Phe (fMLP) and stromal cell-derived factor 1α (SDF-1α) as well as decreased α5β1-integrin-mediated chemoattractant-stimulated adhesion. These GMFG knockdown impaired effects could be reversed by cotransfection of GFP-tagged full-length GMFG. GMFG knockdown cells reduced the cell surface and total protein levels of α5β1-integrin and increased its degradation. Importantly, we demonstrate that GMFG mediates the ubiquitination of β1-integrin through knockdown or overexpression of GMFG. Moreover, GMFG knockdown retarded the efficient recycling of β1-integrin back to the plasma membrane following normal endocytosis of α5β1-integrin, suggesting that the involvement of GMFG in maintaining α5β1-integrin stability may occur in part by preventing ubiquitin-mediated degradation and promoting β1-integrin recycling. Furthermore, we observed that GMFG interacted with syntaxin 4 (STX4) and syntaxin-binding protein 4 (STXBP4); however, only knockdown of STXBP4, but not STX4, reduced monocyte migration and decreased β1-integrin cell surface expression. Knockdown of STXBP4 also substantially inhibited β1-integrin recycling in human monocytes. These results indicate that the effects of GMFG on monocyte migration and adhesion probably occur through preventing ubiquitin-mediated proteasome degradation of α5β1-integrin and facilitating effective β1-integrin recycling back to the plasma membrane.