Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by single molecule imaging techniques

Chemotactic cells can exhibit extreme sensitivity to chemical gradients. Theoretical estimations of the signal inputs required for chemotaxis suggest that the response can be achieved under the strong influence of stochastic input noise generated by the receptors during the transmembrane signaling. This arises a fundamental question regarding the mechanisms for directional sensing: how do cells obtain reliable information regarding gradient direction by using stochastically operating receptors and the downstream molecules? To address this question, we have developed single molecule imaging techniques to visualize signaling molecules responsible for chemotaxis in living Dictyostelium cells, allowing us to monitor the stochastic signaling processes directly. Single molecule imaging of a chemoattractant bound to a receptor demonstrates that signal inputs fluctuate with time and space. Downstream signaling molecules, such as PTEN and a PH domain-containing protein that are constituent parts of chemotactic signaling system, can also be followed at single molecule level in living cells, illuminating the stochastic nature of chemotactic signaling processes. In this report, we start with a brief introduction of chemotactic response of the eukaryotic cells, followed by an explanation for single molecule imaging techniques, and finally discuss these applications to chemotactic signaling system of Dictyostelium cells.     

Chemotaxis in the absence of PIP3 gradients

Chemotaxing neutrophils and Dictyostelium amoebae produce in their plasma membranes the signaling lipid PI(3,4,5)P3 (PIP3) in gradients, which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene-knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is, however, a consistent defect in movement speed in chemotaxis and especially in random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but it can affect cell speed. A signaling pathway from receptor to cytoskeleton able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases must exist.     

Role of phosphatidylinositol 3-kinases in chemotaxis in Dictyostelium

Experiments in several cell types revealed that local accumulation of phosphatidylinositol 3,4,5-triphosphate mediates the ability of cells to migrate during gradient sensing. We took a systematic approach to characterize the functions of the six putative Class I phosphatidylinositol 3-kinases (PI3K1-6) in Dictyostelium by creating a series of gene knockouts. These studies revealed that PI3K1-PI3K3 are the major PI3Ks for chemoattractant-mediated phosphatidylinositol 3,4,5-triphosphate production. We studied chemotaxis of the pi3k1/2/3 triple knock-out strain (pi3k1/2/3 null cells) to cAMP under two distinct experimental conditions, an exponential gradient emitted from a micropipette and a shallow, linear gradient in a Dunn chamber, using four cAMP concentrations ranging over a factor of 10,000. Under all conditions tested pi3k1/2/3 null cells moved slower and had less polarity than wild-type cells. pi3k1/2/3 null cells moved toward a chemoattractant emitted by a micropipette, although persistence was lower than that of wild-type or pi3k1/2 null cells. In shallow linear gradients, pi3k1/2 null cells had greater directionality defects, especially at lower chemoattractant concentrations. Our studies suggest that although PI3K is not essential for directional movement under some chemoattractant conditions, it is a key component of the directional sensing pathway and plays a critical role in linear chemoattractant gradients, especially at low chemoattractant concentrations. The relative importance of PI3K in chemotaxis is also dependent on the developmental stage of the cells. Our data suggest that the output of other signaling pathways suffices to mediate directional sensing when cells perceive a strong signal, but PI3K signaling is crucial for detecting weaker signals.     

An information-theoretic characterization of the optimal gradient sensing response of cells

Many cellular systems rely on the ability to interpret spatial heterogeneities in chemoattractant concentration to direct cell migration. The accuracy of this process is limited by stochastic fluctuations in the concentration of the external signal and in the internal signaling components. Here we use information theory to determine the optimal scheme to detect the location of an external chemoattractant source in the presence of noise. We compute the minimum amount of mutual information needed between the chemoattractant gradient and the internal signal to achieve a prespecified chemotactic accuracy. We show that more accurate chemotaxis requires greater mutual information. We also demonstrate that a priori information can improve chemotaxis efficiency. We compare the optimal signaling schemes with existing experimental measurements and models of eukaryotic gradient sensing. Remarkably, there is good quantitative agreement between the optimal response when no a priori assumption is made about the location of the existing source, and the observed experimental response of unpolarized Dictyostelium discoideum cells. In contrast, the measured response of polarized D. discoideum cells matches closely the optimal scheme, assuming prior knowledge of the external gradient-for example, through prolonged chemotaxis in a given direction. Our results demonstrate that different observed classes of responses in cells (polarized and unpolarized) are optimal under varying information assumptions.     

Dynamics of a chemoattractant receptor in living neutrophils during chemotaxis

Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell's leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutrophils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR-GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR-GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of chemoattractant.     

Chemotaxis with directional sensing during Dictyostelium aggregation

We show that the chemotactic movements of colonies of the starving amoeba Dictyostelium discoideum are driven by a force that depends on both the direction of propagation (directional sensing) of reaction-diffusion chemotactic waves and on the gradient of the concentration of the chemoattractant, solving the chemotactic wave paradox. It is shown that the directional sensing of amoebae is due to the sensitivity of the cells to the time variation of the concentration of the chemoattractant combined with its spatial gradient. It is also shown that chemotaxis exclusively driven by local concentration gradient leads to unstable local motion, preventing cells from aggregation. These findings show that the formation of mounds, which initiate multicellularity in Dictyostelium discoideum, is caused by the sensitivity of the amoebae due to three factors, namely, to the direction of propagation of the chemoattractant, to its spatial gradient, and to the emergence of cAMP “emitting centres”, responsible for the local accumulation of the amoebae.     

Spatiotemporal regulation of Ras activity provides directional sensing

Cells' ability to detect and orient themselves in chemoattractant gradients has been the subject of numerous studies, but the underlying molecular mechanisms remain largely unknown [1]. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium, and inhibition of Ras signaling results in directional migration defects [2]. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2 [2-5]. To investigate the role of Ras in directional sensing, we studied the effect of its misregulation by using cells with disrupted RasGAP activity. We identified an ortholog of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. We show that disruption of nfaA leads to spatially and temporally unregulated Ras activity, causing cytokinesis and chemotaxis defects. By using unpolarized, latrunculin-treated cells, we show that tight regulation of Ras is important for gradient sensing. Together, our findings suggest that Ras is part of the cell's compass and that the RasGAP-mediated regulation of Ras activity affects directional sensing.     

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.     

A Novel Chemotaxis Assay in 3-D Collagen Gels by Time-Lapse Microscopy

The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick''s law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.     

Intracellular Encoding of Spatiotemporal Guidance Cues in a Self-Organizing Signaling System for Chemotaxis in Dictyostelium Cells

Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to move directionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases. It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activates the motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network have been reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues. Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision of polarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for the sensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-time waves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.     

Intracellular Encoding of Spatiotemporal Guidance Cues in a Self-Organizing Signaling System for Chemotaxis in Dictyostelium Cells

Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to move directionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases. It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activates the motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network have been reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues. Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision of polarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for the sensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-time waves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.     

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.     

A stochastic description of Dictyostelium chemotaxis

Chemotaxis, the directed motion of a cell toward a chemical source, plays a key role in many essential biological processes. Here, we derive a statistical model that quantitatively describes the chemotactic motion of eukaryotic cells in a chemical gradient. Our model is based on observations of the chemotactic motion of the social ameba Dictyostelium discoideum, a model organism for eukaryotic chemotaxis. A large number of cell trajectories in stationary, linear chemoattractant gradients is measured, using microfluidic tools in combination with automated cell tracking. We describe the directional motion as the interplay between deterministic and stochastic contributions based on a Langevin equation. The functional form of this equation is directly extracted from experimental data by angle-resolved conditional averages. It contains quadratic deterministic damping and multiplicative noise. In the presence of an external gradient, the deterministic part shows a clear angular dependence that takes the form of a force pointing in gradient direction. With increasing gradient steepness, this force passes through a maximum that coincides with maxima in both speed and directionality of the cells. The stochastic part, on the other hand, does not depend on the orientation of the directional cue and remains independent of the gradient magnitude. Numerical simulations of our probabilistic model yield quantitative agreement with the experimental distribution functions. Thus our model captures well the dynamics of chemotactic cells and can serve to quantify differences and similarities of different chemotactic eukaryotes. Finally, on the basis of our model, we can characterize the heterogeneity within a population of chemotactic cells.     

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.     

Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.     

Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP₃) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by β-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeleton-anchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane.     

Are Growth Factors Chemotactic Agents?

We report the first direct observation of chemotaxis in slowly moving malignant cells. Two sarcoma cell lines of different metastatic potential were used. In a direct-viewing chemotaxis chamber with two concentric wells, the pooled trajectories of highly malignant rat T15 cells were strongly biased toward the outer well which contained platelet-derived and insulin-like growth factors. In individual experiments, however, tile trajectories of the T15 cells showed a significant directional bias which, depending on the cell distribution, sometimes deviated by as much as 170° from the gradient direction. Cells of a less malignant rat line, K2, did not respond to the gradient but a strong K2 response appeared if T15 cells were placed in the outer well along with the growth factors. We conclude that stimulated T15 cells release a chemoattractant, for both T15 and K2 cells, which overrides any chemoattractive effect of the growth factors. These results call into question whether growth factors are ever directly chemotactic in this system and demonstrate the need for direct observation in determining whether any substance is a direct chemoattractant.     

Signaling pathways controlling cell polarity and chemotaxis

Many important biological processes, including chemotaxis (directional cell movement up a chemoattractant gradient), require a clearly established cell polarity and the ability of the cell to respond to a directional signal. Recent advances using Dictyostelium cells and mammalian leukocytes have provided insights into the biochemical and molecular pathways that control chemotaxis. Phosphoinositide 3-kinase plays a central and possibly pivotal role in establishing and maintaining cell polarity by regulating the subcellular localization and activation of downstream effectors that are essential for regulating cell polarity and proper chemotaxis. This review outlines our present understanding of these pathways.     

The Relation of Signal Transduction to the Sensitivity and Dynamic Range of Bacterial Chemotaxis

Complex networks of interacting molecular components of living cells are responsible for many important processes, such as signal processing and transduction. An important challenge is to understand how the individual properties of these molecular interactions and biochemical transformations determine the system-level properties of biological functions. Here, we address the issue of the accuracy of signal transduction performed by a bacterial chemotaxis system. The chemotaxis sensitivity of bacteria to a chemoattractant gradient has been measured experimentally from bacterial aggregation in a chemoattractant-containing capillary. The observed precision of the chemotaxis depended on environmental conditions such as the concentration and molecular makeup of the chemoattractant. In a quantitative model, we derived the chemotactic response function, which is essential to describing the signal transduction process involved in bacterial chemotaxis. In the presence of a gradient, an analytical solution is derived that reveals connections between the chemotaxis sensitivity and the characteristics of the signaling system, such as reaction rates. These biochemical parameters are integrated into two system-level parameters: one characterizes the efficiency of gradient sensing, and the other is related to the dynamic range of chemotaxis. Thus, our approach explains how a particular signal transduction property affects the system-level performance of bacterial chemotaxis. We further show that the two parameters can be derived from published experimental data from a capillary assay, which successfully characterizes the performance of bacterial chemotaxis.     

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96‐well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G‐protein G_iα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low‐ and high‐resolution fluorescence microscopy.