PLA2 and PI3K/PTEN pathways act in parallel to mediate chemotaxis

Directed cell migration involves signaling events that lead to local accumulation of PI(3,4,5)P(3), but additional pathways act in parallel. A genetic screen in Dictyostelium discoideum to identify redundant pathways revealed a gene with homology to patatin-like phospholipase A(2). Loss of this gene did not alter PI(3,4,5)P(3) regulation, but chemotaxis became sensitive to reductions in PI3K activity. Likewise, cells deficient in PI3K activity were more sensitive to inhibition of PLA(2) activity. Deletion of the PLA(2) homolog and two PI3Ks caused a strong defect in chemotaxis and a reduction in receptor-mediated actin polymerization. In wild-type cells, chemoattractants stimulated a rapid burst in an arachidonic acid derivative. This response was absent in cells lacking the PLA(2) homolog, and exogenous arachidonic acid reduced their dependence on PI3K signaling. We propose that PLA(2) and PI3K signaling act in concert to mediate chemotaxis, and metabolites of PLA(2) may be important mediators of the response.     

Essential role of PI3-kinase and phospholipase A2 in Dictyostelium discoideum chemotaxis

Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca(2+). The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP(3) receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP(2) levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca(2+).     

Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration

Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) production, a polarized distribution of PIP(3) was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP(3) or induce cell polarization. Thus, the increase in PIP(3) concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.     

Myosin IIA Dependent Retrograde Flow Drives 3D Cell Migration

Epithelial cell migration is an essential part of embryogenesis and tissue regeneration, yet their migration is least understood. Using our three-dimensional (3D) motility analysis, migrating epithelial cells formed an atypical polarized cell shape with the nucleus leading the cell front and a contractile cell rear. Migrating epithelial cells exerted traction forces to deform both the anterior and posterior extracellular matrix toward the cell body. The cell leading edge exhibited a myosin II-dependent retrograde flow with the magnitude and direction consistent with surrounding network deformation. Interestingly, on a two-dimensional substrate, myosin IIA-deficient cells migrated faster than wild-type cells, but in a 3D gel, these myosin IIA-deficient cells were unpolarized and immobile. In contrast, the migration rates of myosin IIB-deficient cells were similar to wild-type cells. Therefore, myosin IIA, not myosin IIB, is required for 3D epithelial cell migration.     

Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions.     

Phosphatidylinositol 3-kinase in the G protein-coupled receptor-induced chemokinesis and chemotaxis of MDA-MB-468 breast carcinoma cells: a comparison with leukocytes

The polarization of tumor cells and leukocytes into a front end and a rear end is a crucial prerequisite for their autonomous, directed movement. Phosphatidylinositol 3-kinase (PI3K) is assumed to play an important role in this polarization process, whereas the results obtained with different cell types and different migration assays widely vary. Thus, we conducted a comparative study on the role of the PI3K in the locomotor activity and directionality of the migration of tumor cells on the example of MDA-MB-468 breast carcinoma cells in comparison with CTLs and neutrophil granulocytes. We used our well-established, collagen-based, three-dimensional migration assay for the investigation of the chemokinesis and chemotaxis of these cells. Our results show that the role of the PI3K in the regulation of migratory activity is distinct between the investigated cell types: the migration of CTLs and MDA-MB-468 cells was impaired by the inhibition of the PI3K with wortmannin, whereas neutrophil granulocytes were only slightly affected. However, neither cell type was impaired in the ability to respond chemotactically to gradients of ligands to G protein-coupled receptors. Thus, the PI3K contributes to the regulation of migratory activity but not to the directionality of migration of MDA-MB-468 breast carcinoma cells. As a further conclusion with regard to cancer treatment, the PI3K is not a suitable target for the inhibition of metastasis formation, because the migration of leukocytes is also affected, which leads to a dysfunction of the immune defense.     

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.     

EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins

DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.     

PTEN modulates GDNF/RET mediated chemotaxis and branching morphogenesis in the developing kidney

The RET receptor tyrosine kinase is activated by GDNF and controls outgrowth and invasion of the ureteric bud epithelia in the developing kidney. In renal epithelial cells and in enteric neuronal precursor cells, activation of RET results in chemotaxis as Ret expressing cells invade the surrounding GDNF expressing tissue. One potential downstream signaling pathway governing RET mediated chemotaxis may require phosphatidylinositol 3-kinase (PI3K), which generates PI(3,4,5) triphosphate. The PTEN tumor suppressor gene encodes a protein and lipid phosphatase that regulates cell growth, apoptosis and many other cellular processes. PTEN helps regulate cellular chemotaxis by antagonizing the PI3K signaling pathway through dephosphorylation of phosphotidylinositol triphosphates. In this report, we show that PTEN suppresses RET mediated cell migration and chemotaxis in cell culture assays, that RET activation results in asymmetric localization of inositol triphosphates and that loss of PTEN affects the pattern of branching morphogenesis in developing mouse kidneys. These data suggest a critical role for the PI3K/PTEN axis in shaping the pattern of epithelial branches in response to RET activation.     

Asymmetric localization of calpain 2 during neutrophil chemotaxis

Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis.     

Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells

We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.     

Guanylyl cyclase protein and cGMP product independently control front and back of chemotaxing Dictyostelium cells

Chemotaxis of amoeboid cells is driven by actin filaments in leading pseudopodia and actin-myosin filaments in the back and at the side of the cell to suppress pseudopodia. In Dictyostelium, cGMP plays an important role during chemotaxis and is produced predominantly by a soluble guanylyl cyclase (sGC). The sGC protein is enriched in extending pseudopodia at the leading edge of the cell during chemotaxis. We show here that the sGC protein and the cGMP product have different functions during chemotaxis, using two mutants that lose either catalytic activity (sGCDelta cat) or localization to the leading edge (sGCDeltaN). Cells expressing sGCDeltaN exhibit excellent cGMP formation and myosin localization in the back of the cell, but they exhibit poor orientation at the leading edge. Cells expressing the catalytically dead sGCDelta cat mutant show poor myosin localization at the back, but excellent localization of the sGC protein at the leading edge, where it enhances the probability that a new pseudopod is made in proximity to previous pseudopodia, resulting in a decrease of the degree of turning. Thus cGMP suppresses pseudopod formation in the back of the cell, whereas the sGC protein refines pseudopod formation at the leading edge.     

Intrinsic Migratory Properties of Cultured Schwann Cells Based on Single-Cell Migration Assay

The migration of Schwann cells is critical for development of peripheral nervous system and is essential for regeneration and remyelination after nerve injury. Although several factors have been identified to regulate Schwann cell migration, intrinsic migratory properties of Schwann cells remain elusive. In this study, based on time-lapse imaging of single isolated Schwann cells, we examined the intrinsic migratory properties of Schwann cells and the molecular cytoskeletal machinery of soma translocation during migration. We found that cultured Schwann cells displayed three motile phenotypes, which could transform into each other spontaneously during their migration. Local disruption of F-actin polymerization at leading front by a Cytochalasin D or Latrunculin A gradient induced collapse of leading front, and then inhibited soma translocation. Moreover, in migrating Schwann cells, myosin II activity displayed a polarized distribution, with the leading process exhibiting higher expression than the soma and trailing process. Decreasing this front-to-rear difference of myosin II activity by frontal application of a ML-7 or BDM (myosin II inhibitors) gradient induced the collapse of leading front and reversed soma translocation, whereas, increasing this front-to-rear difference of myosin II activity by rear application of a ML-7 or BDM gradient or frontal application of a Caly (myosin II activator) gradient accelerated soma translocation. Taken together, these results suggest that during migration, Schwann cells display malleable motile phenotypes and the extension of leading front dependent on F-actin polymerization pulls soma forward translocation mediated by myosin II activity.     

In chemotaxing fibroblasts, both high-fidelity and weakly biased cell movements track the localization of PI3K signaling

Cell movement biased by a chemical gradient, or chemotaxis, coordinates the recruitment of cells and collective migration of cell populations. During wound healing, chemotaxis of fibroblasts is stimulated by platelet-derived growth factor (PDGF) and certain other chemoattractants. Whereas the immediate PDGF gradient sensing response has been characterized previously at the level of phosphoinositide 3-kinase (PI3K) signaling, the sensitivity of the response at the level of cell migration bias has not yet been studied quantitatively. In this work, we used live-cell total internal reflection fluorescence microscopy to monitor PI3K signaling dynamics and cell movements for extended periods. We show that persistent and properly aligned (i.e., high-fidelity) fibroblast migration does indeed correlate with polarized PI3K signaling; accordingly, this behavior is seen only under conditions of high gradient steepness (>10% across a typical cell length of 50 μm) and a certain range of PDGF concentrations. Under suboptimal conditions, cells execute a random or biased random walk, but nonetheless move in a predictable fashion according to the changing pattern of PI3K signaling. Inhibition of PI3K during chemotaxis is accompanied by loss of both cell-substratum contact and morphological polarity, but after a recovery period, PI3K-inhibited fibroblasts often regain the ability to orient toward the PDGF gradient.     

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.     

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 chemotaxis by the orchestrated activation of Ras, PI3K, and TOR

Directed cell migration and cell polarity are crucial in many facets of biological processes. Cellular motility requires a complex array of signaling pathways, in which orchestrated cross-talk, a feedback loop, and multi-component signaling recur. Almost every signaling molecule requires several regulatory processes to be functionally activated, and a lack of a signaling molecule often leads to chemotaxis defects, suggesting an integral role for each component in the pathway. We outline our current understanding of the signaling event that regulates chemotaxis with an emphasis on recent findings associated with the Ras, PI3K, and target of rapamycin (TOR) pathways and the interplay of these pathways. Ras, PI3K, and TOR are known as key regulators of cellular growth. Deregulation of those pathways is associated with many human diseases, such as cancer, developmental disorders, and immunological deficiency. Recent studies in yeast, mammalian cells, and Dictyostelium discoideum reveal another critical role of Ras, PI3K, and TOR in regulating the actin cytoskeleton, cell polarity, and cellular movement. These findings shed light on the mechanism by which eukaryotic cells maintain cell polarity and directed cell movement, and also demonstrate that multiple steps in the signal transduction pathway coordinately regulate cell motility.     

Temporal and spatial regulation of phosphoinositide signaling mediates cytokinesis

Polarity is a prominent feature of both chemotaxis and cytokinesis. In chemotaxis, polarity is established by local accumulation of PI(3,4,5)P3 at the cell's leading edge, achieved through temporal and spatial regulation of PI3 kinases and the tumor suppressor, PTEN. We find that as migrating D. discoideum cells round up to enter cytokinesis, PI(3,4,5)P3 signaling is uniformly suppressed. Then, as the spindle and cell elongate, PI3 kinases and PTEN move to and function at the poles and furrow, respectively. Cell lines lacking both of these enzymatic activities fail to modulate PI(3,4,5)P3 levels, are defective in cytokinesis, and cannot divide in suspension. The cells continue to grow and duplicate their nuclei, generating large multinucleate cells. Furrows that fail to ingress between nuclei are unable to stably accumulate myosin filaments or suppress actin-filled ruffles. We propose that phosphoinositide-linked circuits, similar to those that bring about asymmetry during cell migration, also regulate polarity in cytokinesis.     

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells

Migratory front-back polarity emerges from the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) on adhesive signaling. We demonstrate here that, during polarization, MIIA and MIIB coordinately promote localized actomyosin bundling, which generates large, stable adhesions that do not signal to Rac and thereby form the cell rear. MIIA formed dynamic actomyosin proto-bundles that mark the cell rear during spreading; it also bound to actin filament bundles associated with initial adhesion maturation in protrusions. Subsequent incorporation of MIIB stabilized the adhesions and actomyosin filaments with which it associated and formed a stable, extended rear. These adhesions did not turn over and no longer signal to Rac. Microtubules fine-tuned the polarity by positioning the front opposite the MIIA/MIIB-specified rear. Decreased Rac signaling in the vicinity of the MIIA/MIIB-stabilized proto-bundles and adhesions was accompanied by the loss of Rac guanine nucleotide exchange factor (GEFs), like βPIX and DOCK180, and by inhibited phosphorylation of key residues on adhesion proteins that recruit and activate Rac GEFs. These observations lead to a model for front-back polarity through local GEF depletion.