Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions

Current models of eukaryotic chemotaxis propose that directional sensing causes localized generation of new pseudopods. However, quantitative analysis of pseudopod generation suggests a fundamentally different mechanism for chemotaxis in shallow gradients: first, pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo; second, in Dictyostelium cells in shallow chemoattractant gradients, pseudopods are made at the same rate whether cells are moving up or down gradients. The location and direction of new pseudopods are random within the range allowed by bifurcation and are not oriented by chemoattractants. Thus, pseudopod generation is controlled independently of chemotactic signalling. Third, directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones. Finally, the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002 affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI(3)K regulates the underlying mechanism of cell movement, rather than control of direction.     

Navigation of Chemotactic Cells by Parallel Signaling to Pseudopod Persistence and Orientation

The mechanism of chemotaxis is one of the most interesting issues in modern cell biology. Recent work shows that shallow chemoattractant gradients do not induce the generation of pseudopods, as has been predicted in many models. This poses the question of how else cells can steer towards chemoattractants. Here we use a new computational algorithm to analyze the extension of pseudopods by Dictyostelium cells. We show that a shallow gradient of cAMP induces a small bias in the direction of pseudopod extension, without significantly affecting parameters such as pseudopod frequency or size. Persistent movement, caused by alternating left/right splitting of existing pseudopodia, amplifies the effects of this bias by up to 5-fold. Known players in chemotactic pathways play contrasting parts in this mechanism; PLA2 and cGMP signal to the cytoskeleton to regulate the splitting process, while PI 3-kinase and soluble guanylyl cyclase mediate the directional bias. The coordinated regulation of pseudopod generation, orientation and persistence by multiple signaling pathways allows eukaryotic cells to detect extremely shallow gradients.     

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.     

Actin polymerization and pseudopod extension during amoeboid chemotaxis

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.     

Mechanisms of amoeboid chemotaxis: an evaluation of the cortical expansion model

In this work we evaluate the cortical expansion model for amoeboid chemotaxis with regard to new information about molecular events in the cytoskeleton following chemotactic stimulation of Dictyostelium amoebae. A rapid upshift in the concentration of chemoattractant can be used to synchronize the motile behavior of a large population of cells. This synchrony presents an opportunity to study the biochemical basis of morphological changes such as pseudopod extension that are required for amoeboid chemotaxis. Changes in the composition and activity of the cytoskeleton following stimulation can be measured with precision and correlated with important morphological changes. Such studies demonstrate that activation of actin nucleation is one of the first and most crucial events in the actin cytoskeleton following stimulation. This activation is followed by incorporation of specific actin cross-linking proteins into the cytoskeleton, which are implicated in the extension of pseudopods and filopods. These results, as well as those from studies with mutants deficient in myosin, indicate that cortical expansion, driven by focal actin polymerization, cross-linking and gel osmotic swelling, is an important force for pseudopod extension. It is concluded that whereas three forces, frontal sliding, tail contraction, and cortical expansion may cooperate to produce amoeboid movement, the cortical expansion model offers the simplest explanation of how focal stimulation with a chemoattractant causes polarized pseudopod extension.     

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.     

Molecular insights into eukaryotic chemotaxis.

Many cells display directed migration toward specific compounds. The best-studied eukaryotic models of chemotaxis are polymorphonuclear leukocytes, which respond to formylated peptides and Dictyostelium amoebas, which respond to extracellular cAMP. In both cell types, chemoattractants bind to surface receptors that contain seven transmembrane domains and interact with G proteins. Some cells, such as fibroblasts, undergo chemotaxis toward compounds whose receptors lack this motif and transmit their signals by other mechanisms. The cytosolic changes elicited by chemoattractants include increased levels of cAMP, cGMP, inositol phosphates, and calcium. These changes are correlated with actin polymerization and other cytoskeletal events that result in preferential extension of pseudopods toward the chemoattractant. Dictyostelium cell lines in which specific genes have been disrupted have demonstrated the necessity of a cAMP receptor (cAR1) and a G protein alpha-subunit (G alpha 2) for responsiveness to cAMP. Other proteins, such as myosin heavy chain and several actin binding proteins, are dispensible although their absence does affect the details of chemotaxis. The disruption of other relevant genes and the genetic reconstitution of chemotaxis in cells lacking crucial proteins should reveal many clues about this complicated and fascinating process.     

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.     

Two phases of actin polymerization display different dependencies on PI(3,4,5)P3 accumulation and have unique roles during chemotaxis

The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of Dictyostelium discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P3/PI(3,4)P2 reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild-type cells but partially restore polarity and chemotactic response in pten- cells. Surprisingly, the fast phase of PI(3,4,5)P3 accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis.     

Making all the right moves: chemotaxis in neutrophils and Dictyostelium

Neutrophils and Dictyostelium discoideum share the ability to migrate directionally in response to external chemoattractant gradients. The binding of chemoattractants to specific receptors that are coupled to heterotrimeric G proteins leads to a wide range of biochemical responses that become highly localized as cells polarize and migrate by chemotaxis. The signaling mechanisms that lead to the predominant polymerization of F-actin at the front of cells for propulsion and to myosin II assembly at the sides to suppress lateral pseudopod formation and at the back for retraction are now beginning to emerge.     

Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation

Actin polymerization occurs in amebae of Dictyostelium discoideum after chemotactic stimulation (Hall, A. L., A. Schlein, and J. Condeelis. 1988. J. Cell. Biochem. 37:285-299). When cells are lysed with Triton X-100 during stimulation, an actin nucleation activity is detected in lysates by measuring the rate of pyrene-labeled actin polymerization. This stimulated nucleation activity is closely correlated with actin polymerization observed in vivo in its kinetics, developmental regulation, and cytochalasin D sensitivity. Actin polymerization is coordinate with pseudopod extension in synchronized populations of cells and is correlated with the accumulation of F actin in pseudopods. The stimulated actin nucleation activity is present in low-speed pellets from Triton lysates (cytoskeletons) within 3 s of stimulation and is stable compared with the nucleation activity of whole cell lysates. Low-speed supernatants contain a reversible inhibitor of the actin nucleation activity that is itself regulated by chemotactic stimulation. Neither activity requires Ca2+ and both are fully expressed in 10 mM EGTA. Fractions containing the inhibitor do not sever actin filaments but do inhibit actin polymerization that is seeded by fragments of purified F actin. These results indicate that chemotactic stimulation of Dictyostelium discoideum generates both an actin-nucleating activity and an actin-polymerization inhibitor, and suggest that the parallel regulation of these two activities leads to the transient phases of actin polymerization observed in vivo. The different compartmentation of these two activities may account for polarized pseudopod extension in gradients of chemoattractant.     

Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.     

Controlled pseudopod extension of human neutrophils stimulated with different chemoattractants

The formation of pseudopods and lamellae after ligation of chemoattractant sensitive G-protein coupled receptors (GPCRs) is essential for chemotaxis. Here, pseudopod extension was stimulated with chemoattractant delivered from a micropipet. The chemoattractant diffusion and convection mass transport were considered, and it is shown that when the delivery of chemoattractant was limited by diffusion there was a strong chemoattractant gradient along the cell surface. The diffusion-limited delivery of chemoattractant from a micropipet allowed for maintaining an almost constant chemoattractant concentration at the leading edge of single pseudopods during their growth. In these conditions, the rate of pseudopod extension was dependent on the concentration of chemoattractant in the pipet delivering chemoattractant. The pseudopod extension induced using micropipets was oscillatory even in the presence of a constant delivery of chemoattractant. This oscillatory pseudopod extension was controlled by activated RhoA and its downstream effector kinase ROCK and was abolished after the inhibition of RhoA activation with Clostridium botulinium C3 exoenzyme (C3) or the blocking of ROCK activation with Y-27632. The ability of the micropipet assay to establish a well-defined chemoattractant distribution around the activated cell over a wide range of molecular weights of the used chemoattractants allowed for comparison of the effect of chemoattractant stimulation on the dynamics of pseudopod growth. Pseudopod growth was stimulated using N-formylated peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)), platelet activating factor (PAF), leukotriene B4 (LTB(4)), C5a anaphylotoxin (C5a), and interleukin-8 (IL-8), which represent the typical ligands for G-protein coupled chemotactic receptors. The dependence of the rate of pseudopod extension on the concentration of these chemoattractants and their equimolar mixture was measured and shown to be similar for all chemoattractants. The inhibition of the activity of phosphoinositide-3 kinase (PI3K) with wortmannin showed that 72%-80% of the rate of pseudopod extension induced with N-formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4 was phosphoinositide-3 kinase-dependent, in contrast to 55% of the rate of pseudopod extension induced with interleukin-8. The dependence of the rate of pseudopod extension on the concentration of individual chemoattractants and their equimolar mixture suggests that there is a common rate-limiting mechanism for the polymerization of cytoskeletal F-actin in the pseudopod region induced by G-protein coupled chemoattractant receptors.     

Ponticulin plays a role in the positional stabilization of pseudopods

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer- assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin- minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin- minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.     

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.     

Temporal and spatial regulation of chemotaxis

The ability to sense and respond to shallow gradients of extracellular signals is remarkably similar in Dictyostelium discoideum amoebae and mammalian leukocytes. Chemoattractant receptors and G proteins are fairly evenly distributed along the cell surface. Receptor occupancy generates local excitatory and global inhibitory processes that balance to control the chemotactic response. Uniform stimuli transiently recruit PI3Ks to, and release PTEN from, the plasma membrane, while gradients of chemoattractant cause the two enzymes to bind to the membrane at the front and back of the cell, respectively. Interference with PI3Ks alters chemotaxis, and disruption of PTEN broadens PI localization and actin polymerization in parallel. Thus, counteracting signals from the upstream elements of the pathway converge to regulate the key enzymes of PI metabolism, localize these lipids, and direct pseudopod formation.     

WASP-interacting protein is important for actin filament elongation and prompt pseudopod formation in response to a dynamic chemoattractant gradient

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.     

Navigating signaling networks: chemotaxis in Dictyostelium discoideum

Studies of chemotaxis in the social amoeba Dictyostelium discoideum have revealed numerous conserved signaling networks that are activated by chemoattractants. In the presence of a uniformly distributed stimulus, these pathways are transiently activated, but in a gradient they are activated persistently and can be localized to either the front or the back of the cell. Recent studies have begun to elucidate how chemoattractant signaling regulates the three main components of chemotaxis: directional sensing, pseudopod extension, and polarization.     

Chemoattractant stimulation of polymorphonuclear leucocyte locomotion

Chemoattractants stimulate both cell locomotion and the orientation of this locomotion (chemotaxis) in polymorphonuclear leukocytes. Cell locomotion is a complex process which includes the coordinated protrusion of cell processes, formation of attachments to the substrate and contraction of the rear of the cell. To understand how chemoattractants regulate this process, it is helpful to dissect the process into components that can be examined separately. Comparison of these components in cells before and after stimulation with chemoattractant provides information about their regulation. In this review we focus on three components: how chemoattractants induce the development of cell polarity; how chemoattractants modulate cytoskeletal components (especially actin) to cause pseudopod protrusion; and how chemoattractant modulation of cell adhesions might contribute to cell locomotion. Spatial and temporal coordination of these and other components of locomotion result in efficient and directed cell movement. Our treatment of these questions is speculative and not comprehensive. We propose simple hypothetical models which can provide the reader with a conceptual framework that integrates the information available.     

Diverse chemotactic responses of Dictyostelium discoideum amoebae in the developing (temporal) and stationary (spatial) concentration gradients of folic acid,cAMP, Ca(2+) and Mg(2+)

The responses of Dictyostelium discoideum amoebae to developing (temporal) and stationary (spatial) gradients of folic acid, cAMP, Ca(2+), and Mg(2+) were studied using the methods of computer-aided image analysis. The results presented demonstrate that the new type of experimental chambers used for the observation of single cells moving within the investigated gradients of chemoattractants permit time lapse recording of single amoebae and determination of the trajectories of moving cells. It was found that, besides folic acid and cAMP (natural chemoattractants for Dictyostelium discoideum amoebae), also extracellular Ca(2+) and Mg(2+) are potent inducers of these cells' chemotaxis, and the amoebae of D. discoideum can respond to various chemoattractants differently. In the positively developing gradients of folic acid, cAMP, Ca(2+), and Mg(2+) oriented locomotion of amoebae directed towards the higher concentration of the tested chemoattractants was observed. However, in the negatively developing (temporal) and stationary linear (spatial) gradients, the univocal chemotaxis of amoebae was recorded only in the case of the Mg(2+) concentration gradient. This demonstrates that amoebae can respond to both developing and stationary gradients, depending upon the nature of the chemoattractant. We also investigated the effects of chosen inhibitors of signalling pathways upon chemotaxis of D. discoideum amoebae in the positively developing (temporal) gradients of tested chemoattractants. Verapamil was found to abolish the chemotaxis of amoebae only in the Ca(2+) gradients. Pertussis toxin suppressed the chemotactic response of cells in the gradients of folic acid and cAMP but did not prevent chemotaxis in those of Ca(2+) and Mg(2+), while quinacrine inhibited chemotaxis in the gradients of folic acid, cAMP, and Ca(2+) but only slightly affected chemotaxis in the Mg(2+) gradient. None of the tested inhibitors causes inhibition of cell random movement, when applied in isotropic solution. Also EDTA and EGTA up to 50 mM concentration did not inhibit locomotion of amoebae in control isotropic solutions.