Rac regulation of chemotaxis and morphogenesis in Dictyostelium

Chemotaxis requires localized F-actin polymerization at the site of the plasma membrane closest to the chemoattractant source, a process controlled by Rac/Cdc42 GTPases. We identify Dictyostelium RacB as an essential mediator of this process. RacB is activated upon chemoattractant stimulation, exhibiting biphasic kinetics paralleling F-actin polymerization. racB null cells have strong chemotaxis and morphogenesis defects and a severely reduced chemoattractant-mediated F-actin polymerization and PAKc activation. RacB activation is partly controlled by the PI3K pathway. pi3k1/2 null cells and wild-type cells treated with LY294002 exhibit a significantly reduced second peak of RacB activation, which is linked to pseudopod extension, whereas a PTEN hypomorph exhibits elevated RacB activation. We identify a RacGEF, RacGEF1, which has specificity for RacB in vitro. racgef1 null cells exhibit reduced RacB activation and cells expressing mutant RacGEF1 proteins display chemotaxis and morphogenesis defects. RacGEF1 localizes to sites of F-actin polymerization. Inhibition of this localization reduces RacB activation, suggesting a feedback loop from RacB via F-actin polymerization to RacGEF1. Our findings provide a critical linkage between chemoattractant stimulation, F-actin polymerization, and chemotaxis in Dictyostelium.     

Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by inhibition of PI3-kinase

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.     

The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.     

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.     

Involvement of the Cytoskeleton in Controlling Leading-Edge Function during Chemotaxis

In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders, with activity peaking at ∼3 s after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, as well as elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading-edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.     

Phosphoinositide 3-kinase activity controls the chemoattractant-mediated activation and adaptation of adenylyl cyclase

The binding of chemoattractants to cognate G protein-coupled receptors activates a variety of signaling cascades that provide spatial and temporal cues required for chemotaxis. When subjected to uniform stimulation, these responses are transient, showing an initial peak of activation followed by a period of adaptation, in which activity subsides even in the presence of stimulus. A tightly regulated balance between receptor-mediated stimulatory and inhibitory pathways controls the kinetics of activation and subsequent adaptation. In Dictyostelium, the adenylyl cyclase expressed during aggregation (ACA), which synthesizes the chemoattractant cAMP, is essential to relay the signal to neighboring cells. Here, we report that cells lacking phosphoinositide 3-kinase (PI3K) activity are deficient in signal relay. In LY294002-treated cells, this defect is because of a loss of ACA activation. In contrast, in cells lacking PI3K1 and PI3K2, the signal relay defect is because of a loss of ACA adaptation. We propose that the residual low level of 3-phosphoinositides in pi3k(1-/2-) cells is sufficient to generate the initial peak of ACA activity, yet is insufficient to sustain the inhibitory phase required for its adaptation. Thus, PI3K activity is poised to regulate both ACA activation and adaptation, thereby providing a link to ensure the proper balance of counteracting signals required to maintain optimal chemoresponsiveness.     

Tumor suppressor PTEN mediates sensing of chemoattractant gradients

Shallow gradients of chemoattractants, sensed by G protein-linked signaling pathways, elicit localized binding of PH domains specific for PI(3,4,5)P3 at sites on the membrane where rearrangements of the cytoskeleton and pseudopod extension occur. Disruption of the PI 3-phosphatase, PTEN, in Dictyostelium discoideum dramatically prolonged and broadened the PH domain relocation and actin polymerization responses, causing the cells lacking PTEN to follow a circuitous route toward the attractant. Exogenously expressed PTEN-GFP localized to the surface membrane at the rear of the cell. Membrane localization required a putative PI(4,5)P2 binding motif and was required for chemotaxis. These results suggest that specific phosphoinositides direct actin polymerization to the cell's leading edge and regulation of PTEN through a feedback loop plays a critical role in gradient sensing and directional migration.     

Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells

Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the membrane at the front and back, respectively, of chemotaxing Dictyostelium discoideum cells. However, the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon chemoattractant stimulation, and the changes are greater and more prolonged in pten- cells. PI3K activation increases within 5 s of chemoattractant addition and then declines to a low level of activity identically in wild-type and pten- cells. Reconstitution of the PI3K activation profile can be achieved by mixing membranes from stimulated pi3k1-/pi3k2- cells with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate to shape the temporal and spatial localization of PI(3,4,5)P3.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients

Chemotaxis plays a central role in various biological processes, such as the movement of neutrophils and macrophage during wound healing and in the aggregation of Dictyostelium cells. During the past few years, new understanding of the mechanisms controlling chemotaxis has been obtained through molecular genetic and biochemical studies of Dictyostelium and other experimental systems. This review outlines our present understanding of the signaling pathways that allow a cell to sense and respond to a chemoattractant gradient. In response to chemoattractants, cells either become polarized in the direction of the chemoattractant source, which results in the formation of a leading edge, or they reorient their polarity in the direction of the chemoattractant gradient and move with a stronger persistence up the gradient. Models are presented here to explain such directional responses. They include a localized activation of pathways at the leading edge and an "inhibition" of these pathways along the lateral edges of the cell. One of the primary pathways that may be responsible for such localized responses is the activation of phosphatidyl inositol-3 kinase (PI3K). Evidence suggests that a localized formation of binding sites for PH (pleckstrin homology) domain-containing proteins produced by PI3K leads to the formation of "activation domains" at the leading edge, producing a localized response.     

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P(3), the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P(3) asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.     

Signal transduction in neutrophil chemotaxis

This review discusses current knowledge on signal transduction pathways controlling chemotaxis of neutrophils and similar cells. Most neutrophil chemoattractants bind to seven-transmembrane-helix receptors. These receptors activate trimeric G proteins of the Gi class in neutrophils to initiate chemotaxis. Phospholipases Cbeta, phosphoinositide 3-kinase gamma, and PH domain-containing proteins play various roles in signaling further downstream. The actin cytoskeleton is crucial for cell motility, and is controlled by Rho family GTP-binding proteins. PIP 5-kinase, LIM kinase, myosin light chain kinase and phosphatase, or WASP-like proteins may be important links between Rho GTPases and actin during chemotaxis. Newly emerging ideas on the regulation of the "compass" of chemotaxing cells, which may involve Cdc42 and certain PH domain-containing proteins, are also presented.     

A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

Increased intracellular H(+) efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1(-)) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1(-) cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI((3,4,5))P(3)). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI((3,4,5))P(3) production and for attaining a polarized phenotype.     

Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa

We demonstrate that PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, which is required for myosin II assembly. We demonstrate that PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis.     

Regulation of protein kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines

Protein Kinase B (PKB/Akt) is a key regulator of cell proliferation, motility and survival. The activation status of PKB is regulated by phosphatidylinositol 3-kinase (PI3K) via the synthesis of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3, PIP3). PTEN antagonises PI3K by degrading PIP3 to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Deregulation of PKB through loss of functional PTEN has frequently been implicated in the progression of tumours, including prostate cancer, and the PTEN-negative prostate cancer cell lines LNCaP and PC3 have been widely used as models for this mechanism of constitutive PKB activation. However, other enzymes in addition to PTEN can antagonise PI3K, including SHIP2, which degrades PIP3 to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). We investigated the role of PTEN and SHIP2 in the regulation of PKB phosphorylation in a panel of human prostate-derived epithelial cell lines. In the PTEN-positive prostate-derived cell lines PNT2, PNT1a and P4E6, PI3K inhibition by LY294002 caused rapid dephosphorylation of PKB at ser473 (T(1/2)<2 min), leading to its inactivation. In the PTEN-null line LNCaP, LY294002-induced PKB dephosphorylation was much slower (T(1/2)>20 min), but in PC3 cells (also PTEN-null) it was only slightly slower than in PTEN-positive cells (T(1/2)=3 min). PKB dephosphorylation paralleled loss of plasma membrane PIP3. PNT1a, P4E6 and PC3, but not PNT2 or LNCaP, expressed SHIP2. SiRNA-mediated knockdown of SHIP2 expression markedly slowed PKB inactivation in response to LY294002 in PC3 but not in other SHIP2-positive cells, whereas knockdown of PTEN expression in PNT2, PNT1a and P4E6 resulted in higher steady-state levels of PKB phosphorylation and slowed, but did not prevent, LY294002-induced PKB inactivation. Thus SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells but not other prostate cell lines, where PTEN may share this role with further PIP3-degrading mechanisms.     

PDGF stimulates DNA synthesis in human vascular smooth muscle cells via a novel wortmannin-insensitive phosphatidylinositol 3-kinase

The class 1(A) phosphatidylinositol 3-kinase enzymes consist of a number of heterodimeric complexes of regulatory and catalytic subunits and have been implicated in a number of cellular responses. While platelet-derived growth factor (PDGF)-induced chemotaxis of human vascular smooth muscle cells (HVSMC) is inhibited by both wortmannin and LY294002, DNA synthesis is only inhibited by LY294002. Serum-induced DNA synthesis however is inhibited by LY294002, wortmannin and rapamycin. Similarly PDGF-induced protein kinase B (PKB) activation is inhibited by LY294002 but not by wortmannin or rapamycin. In conclusion PDGF-induced DNA synthesis appears to occur through a phosphatidylinositol 3-kinase (PI3-K)-dependent, but wortmannin-insensitive, PKB/Akt pathway.     

Phospholipase C regulation of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.     

Genetic analysis of the role of G protein-coupled receptor signaling in electrotaxis

Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.     

Role of RacC for the regulation of WASP and phosphatidylinositol 3-kinase during chemotaxis of Dictyostelium

WASP family proteins are key players for connecting multiple signaling pathways to F-actin polymerization. To dissect the highly integrated signaling pathways controlling WASP activity, we identified a Rac protein that binds to the GTPase binding domain of WASP. Using two-hybrid and FRET-based functional assays, we identified RacC as a major regulator of WASP. RacC stimulates F-actin assembly in cell-free systems in a WASP-dependent manner. A FRET-based microscopy approach showed local activation of RacC at the leading edge of chemotaxing cells. Cells overexpressing RacC exhibit a significant increase in the level of F-actin polymerization upon cAMP stimulation, which can be blocked by a phosphatidylinositol (PI) 3-kinase inhibitor. Membrane translocation of PI 3-kinase and PI 3,4,5-trisphosphate reporter is absent in racC null cells. Cells overexpressing dominant negative RacC mutants and racC null cells move at a significantly slower speed and show a poor directionality during chemotaxis. Our results suggest that RacC plays an important role in PI 3-kinase activation and WASP activation for dynamic regulation of F-actin assembly during Dictyostelium chemotaxis.     

The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.