Role of modulators of small GTPases in chemotaxis, cytokinesis and development in Dictyostelium Discoideum

The work described here shows the complexity of GTPase signalling in an apparently simple organism Dictyostelium discoideum. Ras Guanine nucleotide exchange factor RasGEF Q is one out of at least 25 RasGEFs in D. discoideum. Here we show that it specifically regulates myosin II functions by regulating myosin phosphorylation. RasGEF Q activates the Ras isoform RasB upon stimulation with cAMP. Activated RasB can directly or indirectly activate Myosin Heavy chain kinase A (MHCK A) which then phosphorylates the myosin II heavy chain. Phosphorylated myosin II cannot assemble into filaments and is thus a non-functional form. Furthermore, a DEP domain in RasGEF Q appears to be critical for activating RasGEF Q by release from an autoinhibited state. Studies on the multidomain Rac Guanine nucleotide exchange factor GxcDD show that it is required for the early phases of development in chemotactic migration and streaming behaviour. The characterization of its single domains revealed that the CH domain (Calponin homology domain) of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases and the ArfGAP-PH tandem accumulates in cortical regions of the cell and in phagosomes. Our results also suggest that a conformational change is required for activation of GxcDD, which would be important for its downstream signaling. Studies on the IQGAP related protein GAPA showed that it associates with two actin-corsslinking protein, namely Filamin and Cortexillin I. GAPA is required for cytokinesis and localizes to the cleavage furrow during cytokinesis in a cortexillin I dependent way. We also observed that GAPA is required for proper phototaxis as is its binding partner Filamin

GxcDD, a putative RacGEF, is involved in Dictyostelium development

<p>Abstract</p> <p>Background</p> <p>Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba <it>Dictyostelium discoideum </it>lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.</p> <p>Results</p> <p>We report on GxcDD (<b>G</b>uanine e<b>x</b>change factor for Ra<b>c </b>GTPases), a <it>Dictyostelium </it>RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling.</p> <p>Conclusion</p> <p>The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in <it>Dictyostelium</it>, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further.</p

Phosphoinositide Lipid Posphatase SHIP1 and PTEN Coordinate to Regulate Cell Migration and Adhesion

The second messenger phosphatidylinositol$(3,4,5)P_3 (PtdIns(3,4,5)P_3)$ is formed by stimulation of various receptors, including G protein–coupled receptors and integrins. The lipid phosphatases PTEN and SHIP1 are critical in regulating the level of PtdIns$(3,4,5)P_3$ during chemotaxis. Observations that loss of PTEN had minor and loss of SHIP1 resulted in a severe chemotaxis defect in neutrophils led to the belief that SHIP1 rather than PTEN acts as a predominant phospholipid phosphatase in establishing a PtdIns$(3,4,5)P_3$ compass. In this study, we show that SHIP1 regulates PtdIns$(3,4,5)P_3$ production in response to cell adhesion and plays a limited role when cells are in suspension. $SHIP1^{−/−}$ neutrophils lose their polarity upon cell adhesion and are extremely adherent, which impairs chemotaxis. However, chemo­taxis can be restored by reducing adhesion. Loss of SHIP1 elevates Akt activation following cell adhesion due to increased PtdIns$(3,4,5)P_3$ production. From our observations, we conclude that SHIP1 prevents formation of top-down PtdIns$(3,4,5)P_3$ polarity to facilitate proper cell attachment and detachment during chemotaxis

Linking Ras to myosin function: RasGEF Q, a Dictyostelium exchange factor for RasB, affects myosin II functions

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q− mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q− mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II

Profilin isoforms in Dictyostelium discoideum

AbstractEukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform

Guanine Nucleotide Exchange Factor Vav1 Regulates Perivascular Homing and Bone Marrow Retention of Hematopoietic Stem and Progenitor Cells

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted $Vav1^{−/−}$ HSPC showed impaired early localization near $nestin^+$ perivascular mesenchymal stem cells; only 6.25% of $Vav1^{−/−}$ HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a $nestin^+$ cell. Abnormal perivascular localization correlated with decreased retention of $Vav1^{−/−}$ HSPC in the bone marrow (44–60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of $Vav1^{−/−}$ cells vs. 53–63% for wild-type cells). The engraftment defect of $Vav1^{−/−}$ HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, $Vav1^{−/−}$ HSPC showed impaired responses to $SDF1\alpha$, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for $SDF1\alpha$-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment

Myeloid Cell-Derived Reactive Oxygen Species Externally Regulate the Proliferation of Myeloid Progenitors in Emergency Granulopoiesis

SummaryThe cellular mechanisms controlling infection-induced emergency granulopoiesis are poorly defined. Here we found that reactive oxygen species (ROS) concentrations in the bone marrow (BM) were elevated during acute infection in a phagocytic NADPH oxidase-dependent manner in myeloid cells. Gr1+ myeloid cells were uniformly distributed in the BM, and all c-kit+ progenitor cells were adjacent to Gr1+ myeloid cells. Inflammation-induced ROS production in the BM played a critical role in myeloid progenitor expansion during emergency granulopoiesis. ROS elicited oxidation and deactivation of phosphatase and tensin homolog (PTEN), resulting in upregulation of PtdIns(3,4,5)P3 signaling in BM myeloid progenitors. We further revealed that BM myeloid cell-produced ROS stimulated proliferation of myeloid progenitors via a paracrine mechanism. Taken together, our results establish that phagocytic NADPH oxidase-mediated ROS production by BM myeloid cells plays a critical role in mediating emergency granulopoiesis during acute infection

Small Molecule-Induced Cytosolic Activation of Protein Kinase Akt Rescues Ischemia-Elicited Neuronal Death

Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions

G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling

Cytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation

Regulation of the Actin Cytoskeleton by an Interaction of IQGAP Related Protein GAPA with Filamin and Cortexillin I

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin