cAMP Gradients Research Articles

PakD, a Putative p21-Activated Protein Kinase in Dictyostelium discoideum, Regulates Actin

Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum. We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD(-) cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD(-) cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD(-) cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD(-) cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

An attenuating role of a WASP-related protein, WASP-B, in the regulation of F-actin polymerization and pseudopod formation via the regulation of RacC during Dictyostelium chemotaxis

The WASP family of proteins has emerged as important regulators that connect multiple signaling pathways to regulate the actin cytoskeleton. Dictyostelium cells express WASP, as well as a WASP related protein, WASP-B, endoded by wasB gene. WASP-B contains many of the domains present in WASP. Analysis of wild type, wasB null cells revealed that WASP-B is required for proper control of F-actin polymerization in response to a cAMP gradient. Due to the lack of tight control on actin polymerization, wasB null cells exhibited higher level of F-actin polymerization. wasB− cells extend more de novo pseudopods laterally and their average life span is longer than those of wild type cells, causing more turns and inefficient chemotaxis. YFP-WASP-B appears to be uniformly distributed in the cytosol and shows no translocation to cortical membrane upon cAMP stimulation. Active RacC pull-down assay reveals that the level of active RacC in wasB− cells is significantly higher than wild type cells. Moreover, the distribution of active RacC is not localized in wasB− cells. We conclude that chemotaxis defects of wasB− cells are likely to result from the aberrant regulation of RacC activation and localization.

Phosphorylation of Actin-related Protein 2 (Arp2) Is Required for Normal Development and cAMP Chemotaxis in Dictyostelium

Phosphorylation of the actin-related protein 2 (Arp2) subunit of the Arp2/3 complex on evolutionarily conserved threonine and tyrosine residues was recently identified and shown to be necessary for nucleating activity of the Arp2/3 complex and membrane protrusion of Drosophila cells. Here we use the Dictyostelium diploid system to replace the essential Arp2 protein with mutants that cannot be phosphorylated at Thr-235/6 and Tyr-200. We found that aggregation of the resulting mutant cells after starvation was substantially slowed with delayed early developmental gene expression and that chemotaxis toward a cAMP gradient was defective with loss of polarity and attenuated F-actin assembly. Chemotaxis toward cAMP was also diminished with reduced cell speed and directionality and shorter pseudopod lifetime when Arp2 phosphorylation mutant cells were allowed to develop longer to a responsive state similar to that of wild-type cells. However, clathrin-mediated endocytosis and chemotaxis under agar to folate in vegetative cells were only subtly affected in Arp2 phosphorylation mutants. Thus, phosphorylation of threonine and tyrosine is important for a subset of the functions of the Arp2/3 complex, in particular an unexpected major role in regulating development.

Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium

Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0,1-1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced typical for attractant action the increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.

Regulation of multiple tip formation by caffeine in cellular slime molds.

BackgroundThe multicellular slug in Dictyostelium has a single tip that acts as an organising centre patterning the rest of the slug. High adenosine levels at the tip are believed to be responsible for this tip dominance and the adenosine antagonist, caffeine overrides this dominance promoting multiple tip formation.ResultsCaffeine induced multiple tip effect is conserved in all the Dictyostelids tested. Two key components of cAMP relay namely, cAMP phosphodiesterase (Pde4) and adenyl cyclase-A (AcaA) levels get reduced during secondary tip formation in Dictyostelium discoideum. Pharmacological inhibition of cAMP phosphodiesterase also resulted in multiple tips. Caffeine reduces cAMP levels by 16.4, 2.34, 4.71 and 6.30 folds, respectively in D. discoideum, D. aureostipes, D. minutum and Polysphondylium pallidum. We propose that altered cAMP levels, perturbed cAMP gradient and impaired signalling may be the critical factors for the origin of multiple tips in other Dictyostelids as well. In the presence of caffeine, slug cell movement gets impaired and restricted. The cell type specific markers, ecmA (prestalk) and pspA (prespore) cells are not equally contributing during additional tip formation. During additional tip emergence, prespore cells transdifferentiate to compensate the loss of prestalk cells.ConclusionCaffeine decreases adenyl cyclase–A (AcaA) levels and as a consequence low cAMP is synthesised altering the gradient. Further if cAMP phosphodiesterase (Pde4) levels go down in the presence of caffeine, the cAMP gradient breaks down. When there is no cAMP gradient, directional movement is inhibited and might favour re-differentiation of prespore to prestalk cells.

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.

Impact of the carbazole derivative wiskostatin on mechanical stability and dynamics of motile cells

Many essential functions in eukaryotic cells like phagocytosis, division, and motility rely on the dynamical properties of the actin cytoskeleton. A central player in the actin system is the Arp2/3 complex. Its activity is controlled by members of the WASP (Wiskott-Aldrich syndrome protein) family. In this work, we investigated the effect of the carbazole derivative wiskostatin, a recently identified N-WASP inhibitor, on actin-driven processes in motile cells of the social ameba Dictyostelium discoideum. Drug-treated cells exhibited an altered morphology and strongly reduced pseudopod formation. However, TIRF microscopy images revealed that the overall cortical network structure remained intact. We probed the mechanical stability of wiskostatin-treated cells using a microfluidic device. While the total amount of F-actin in the cells remained constant, their stiffness was strongly reduced. Furthermore, wiskostatin treatment enhanced the resistance to fluid shear stress, while spontaneous motility as well as chemotactic motion in gradients of cAMP were reduced. Our results suggest that wiskostatin affects the mechanical integrity of the actin cortex so that its rigidity is reduced and actin-driven force generation is impaired.

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

The IplA Ca++ Channel OfDictyostelium discoideumIs Necessary For Ca++, But Not cAMP Chemotaxis, And Plays A Fundamental Role In Natural Aggregation

During aggregation of Dictyostelium discoideum, nondissipating, symmetrical, outwardly moving waves of cAMP direct cells towards aggregation centers. It has been assumed that the spatial and temporal characteristics of the front and back of each cAMP wave regulate both chemokinesis and chemotaxis. However, during the period preceding aggregation, cells acquire not only the capacity to chemotax in a spatial gradient of cAMP, but also in a spatial gradient of Ca(2+). The null mutant of the putative IplA Ca(2+) channel gene, iplA(-), undergoes normal chemotaxis in spatial gradients of cAMP and normal chemokinetic responses to increasing temporal gradients of cAMP, both generated in vitro. However, iplA(-) cells lose the capacity to undergo chemotaxis in response to a spatial gradient of Ca(2+), suggesting that IplA is either the Ca(2+) chemotaxis receptor or an essential component of the Ca(2+) chemotaxis regulatory pathway. In response to natural chemotactic waves generated by wild-type cells, the chemokinetic response of iplA(-) cells to the temporal dynamics of the cAMP wave is intact, but the capacity to reorient in the direction of the aggregation center at the onset of each wave is lost. These results suggest that transient Ca(2+) gradients formed between cells at the onset of each natural cAMP wave augment reorientation towards the aggregation center. If this hypothesis proves correct, it will provide a more complex contextual framework for interpreting D. discoideum chemotaxis.

Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses

Dictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P3 responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding to cAMP. Taken together, these findings suggest that similar components control gradient sensing during FA- and cAMP-mediated motility, but the response of polarized cells is more stable, which ultimately helps maintain their directionality.

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum

Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of Dictyostelium discoideum revealed that all play a minor role in the efficiency of basic cell motility, but none play a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, the two kinases MhckA and MhckC were essential for chemotaxis in a spatial gradient of Ca(2+), shear-induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The phenotypes of the mutants mhckA(-) and mhckC(-) were highly similar to that of the Ca(2+) channel/receptor mutant iplA(-) and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca(2+), but not cAMP, and suggest that Ca(2+) chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.

Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells

Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.

Dictyostelium chemotaxis: essential Ras activation and accessory signalling pathways for amplification

Central to chemotaxis is the molecular mechanism by which cells exhibit directed movement in shallow gradients of a chemoattractant. We used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signalling module providing activation of Ras at the leading edge, which is sufficient for chemotaxis. The signalling enzymes PI3K, TorC2, PLA2 and sGC are not required for Ras activation and chemotaxis to folate or to steep gradients of cAMP, but they provide a memory of direction and improved orientation of the cell, which together increase the sensitivity about 150-fold for chemotaxis in shallow cAMP gradients.

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of <em>Dictyostelium Discoideum</em>

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly (1). This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells (2, 3). A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo (4). The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells (5-8). The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits (7, 9, 10). Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP(2;) into PIP(3;) on the cell membrane (11-13). PIP(3;) serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane (14, 15). Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP(3;) to PIP(2;)(16, 17). The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils (18). We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP(3;) responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of <em>Dictyostelium Discoideum</em>

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits 7, 9, 10. Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

How uniform is cAMP signaling? Focus on “Systems analysis of GLP-1 receptor signaling in pancreatic β-cells”

the goal of many cell physiologists is to ascribe cellular behavior to specific molecular signaling mechanisms. However, the reductionist experimental approach commonly used to understand molecular function often makes this task harder rather than easier to accomplish because of the complexity of

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.

Transient Increase in Cyclic AMP Localized to Macrophage Phagosomes

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients of cAMP is increasingly appreciated. Previous work in macrophages has shown that cAMP is produced during phagocytosis and that elevated cAMP levels suppress host defense functions, including generation of proinflammatory mediators, phagocytosis and killing. However, the spatial and kinetic characteristics of cAMP generation in phagocytosing macrophages have yet to be examined. Using a Förster resonance energy transfer (FRET)-based cAMP biosensor, we measured the generation of cAMP in live macrophages. We detected no difference in bulk intracellular cAMP levels between resting cells and cells actively phagocytosing IgG-opsonized particles. However, analysis with the biosensor revealed a rapid decrease in FRET signal corresponding to a transient burst of cAMP production localized to the forming phagosome. cAMP levels returned to baseline after the particle was internalized. These studies indicate that localized increases in cAMP accompany phagosome formation and provide a framework for a more complete understanding of how cAMP regulates macrophage host defense functions.

A Stochastic Model for Chemotaxis Based on the Ordered Extension of Pseudopods

Many amoeboid cells move by extending pseudopods. Here I present a new stochastic model for chemotaxis that is based on pseudopod extensions by Dictyostelium cells. In the absence of external cues, pseudopod extension is highly ordered with two types of pseudopods: de novo formation of a pseudopod at the cell body in random directions, and alternating right/left splitting of an existing pseudopod that leads to a persistent zig-zag trajectory. We measured the directional probabilities of the extension of splitting and de novo pseudopods in chemoattractant gradients with different steepness. Very shallow cAMP gradients can bias the direction of splitting pseudopods, but the bias is not perfect. Orientation of de novo pseudopods require much steeper cAMP gradients and can be more precise. These measured probabilities of pseudopod directions were used to obtain an analytical model for chemotaxis of cell populations. Measured chemotaxis of wild-type cells and mutants with specific defects in these stochastic pseudopod properties are similar to predictions of the model. These results show that combining splitting and de novo pseudopods is a very effective way for cells to obtain very high sensitivity to stable gradient and still be responsive to changes in the direction of the gradient.

Ca2+ chemotaxis inDictyostelium discoideum

Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca(2+) functions as a chemoattractant of aggregation-competent Dictyostelium discoideum amoebae, that parallel spatial gradients of cAMP and Ca(2+) are more effective than either alone, and that cAMP functions as a stronger chemoattractant than Ca(2+). Effective Ca(2+) gradients are extremely steep compared with effective cAMP gradients. This presents a paradox because there is no indication to date that steep Ca(2+) gradients are generated in aggregation territories. However, given that Ca(2+) chemotaxis is co-acquired with cAMP chemotaxis during development, we speculate on the role that Ca(2+) chemotaxis might have and the possibility that steep, transient Ca(2+) gradients are generated during natural aggregation in the interstitial regions between cells.