Oscillatory Behavior of Neutrophils under Opposing Chemoattractant Gradients Supports a Winner-Take-All Mechanism

Neutrophils constitute the largest class of white blood cells and are the first responders in the innate immune response. They are able to sense and migrate up concentration gradients of chemoattractants in search of primary sites of infection and inflammation through a process known as chemotaxis. These chemoattractants include formylated peptides and various chemokines. While much is known about chemotaxis to individual chemoattractants, far less is known about chemotaxis towards many. Previous studies have shown that in opposing gradients of intermediate chemoattractants (interleukin-8 and leukotriene B₄), neutrophils preferentially migrate toward the more distant source. In this work, we investigated neutrophil chemotaxis in opposing gradients of chemoattractants using a microfluidic platform. We found that primary neutrophils exhibit oscillatory motion in opposing gradients of intermediate chemoattractants. To understand this behavior, we constructed a mathematical model of neutrophil chemotaxis. Our results suggest that sensory adaptation alone cannot explain the observed oscillatory motion. Rather, our model suggests that neutrophils employ a winner-take-all mechanism that enables them to transiently lock onto sensed targets and continuously switch between the intermediate attractant sources as they are encountered. These findings uncover a previously unseen behavior of neutrophils in opposing gradients of chemoattractants that will further aid in our understanding of neutrophil chemotaxis and the innate immune response. In addition, we propose a winner-take-all mechanism allows the cells to avoid stagnation near local chemical maxima when migrating through a network of chemoattractant sources.     

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and--unexpectedly--lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractants.     

Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.     

Residence time calculation for chemotactic bacteria within porous media

Local chemical gradients can have a significant impact on bacterial population distributions within subsurface environments by evoking chemotactic responses. These local gradients may be created by consumption of a slowly diffusing nutrient, generation of a local food source from cell lysis, or dissolution of nonaqueous phase liquids trapped within the interstices of a soil matrix. We used a random walk simulation algorithm to study the effect of a local microscopic gradient on the swimming behavior of bacteria in a porous medium. The model porous medium was constructed using molecular dynamics simulations applied to a fluid of equal-sized spheres. The chemoattractant gradient was approximated with spherical symmetry, and the parameters for the swimming behavior of soil bacterium Pseudomonas putida were based on literature values. Two different mechanisms for bacterial chemotaxis, one in which the bacteria responded to both positive and negative gradients, and the other in which they responded only to positive gradients, were compared. The results of the computer simulations showed that chemotaxis can increase migration through a porous medium in response to microscopic-scale gradients. The simulation results also suggested that a more significant role of chemotaxis may be to increase the residence time of the bacteria in the vicinity of an attractant source.     

Integrating conflicting chemotactic signals. The role of memory in leukocyte navigation.

Leukocytes navigate through complex chemoattractant arrays, and in so doing, they must migrate from one chemoattractant source to another. By evaluating directional persistence and chemotaxis during neutrophil migration under agarose, we show that cells migrating away from a local chemoattractant, against a gradient, display true chemotaxis to distant agonists, often behaving as if the local gradient were without effect. We describe two interrelated properties of migrating cells that allow this to occur. First, migrating leukocytes can integrate competing chemoattractant signals, responding as if to the vector sum of the orienting signals present. Second, migrating cells display memory of their recent environment: cells' perception of the relative strength of orienting signals is influenced by their history, so that cells prioritize newly arising or newly encountered attractants. We propose that this cellular memory, by promoting sequential chemotaxis to one attractant after another, is in fact responsible for the integration of competitive orienting signals over time, and allows combinations of chemoattractants to guide leukocytes in a step-by-step fashion to their destinations within tissues.     

“Pseudochemotaxis” by micro-organisms in an attractant gradient

The random behavior of microorganisms in defined attractant gradients may exhibit apparent chemotaxis, or pseudochemotaxis, if the random motility of the population is a function of the attractant concentration. Several examples are studied using computer techniques to solve the differential equations of motion for populations of micro-organisms responding chemokinetically to variable attractant concentrations. The chemokinetic coefficient is a convenient parameter to characterize the time course of experiments in which micro-organisms move in attractant gradients. Measurements of the chemokinetic coefficient as a function of time may be used to directly determine the random mobility as a function of attractant concentration.     

How do leucocytes perceive chemical gradients?

Abstract Chemoattractants determine not only the direction of leucocyte locomotion (chemotaxis) but also its speed (chemokinesis). Various mechanisms by which leucocytes may detect chemotactic gradients, including spatial and temporal detection, are briefly reviewed. These mechanisms as originally proposed did not address the question how attractants cause leucocytes to migrate in persistent random paths in the absence of a gradient. Stochastic models have recently been presented in which leucocytes either respond by polarizing and migrating in the direction from which they receive their first signal, or respond to random flucuations in the perceived attractant concentration. Stochastic models allow an explanation for the persistent random walk shown by cells in uniform concentrations of attractant as well as for directional locomotion in gradients. They suggest that, at the biochemical level, the mechanisms by which attractants stimulate chemotaxis and chemokinesis are probably the same.     

How do leucocytes perceive chemical gradients?

Chemoattractants determine not only the direction of leucocyte locomotion (chemotaxis) but also its speed (chemokinesis). Various mechanisms by which leucocytes may detect chemotactic gradients, including spatial and temporal detection, are briefly reviewed. These mechanisms as originally proposed did not address the question how attractants cause leucocytes to migrate in persistent random paths in the absence of a gradient. Stochastic models have recently been presented in which leucocytes either respond by polarizing and migrating in the direction from which they receive their first signal, or respond to random fluctuations in the perceived attractant concentration. Stochastic models allow an explanation for the persistent random walk shown by cells in uniform concentrations of attractant as well as for directional locomotion in gradients. They suggest that, at the biochemical level, the mechanisms by which attractants stimulate chemotaxis and chemokinesis are probably the same.     

Signal perception of fibroblasts for directional migration to platelet-derived growth factor in Boyden-type chambers

Fibroblast chemotaxis has usually been determined in Boyden-type chambers with polycarbonate filters, assuming that a stable concentration gradient of the attractant develops that causes directional migration of the cells. This view has been repeatedly challenged, and development of such gradients in vivo is unlikely. The present experiments were designed to test if a stable concentration gradient was required for normal dermal fibroblasts to migrate toward platelet-derived growth factor. It was found that a brief pulse of the attractant was required and sufficient to induce chemotaxis. The pulse had to contain a specific concentration of attractant and was ineffective when not unilateral. The observed effects could not be attributed to induction of random migration or migration on a mediator-coated surface. It is not clear which machinery is regulating this cellular behaviour, but it is suggested that cells may migrate in vivo by similar mechanisms, because the establishment of stable concentration gradients of attractants in tissues is deemed unlikely.     

Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.     

Measurement of the chemotaxis coefficient for human neutrophils in the under-agarose migration assay

Clinical and scientific investigations of leukocyte chemotaxis will be greatly aided by an ability to measure quantitative parameters characterizing the intrinsic random motility, chemokinetic, and chemotactic properties of cell populations responding to a given attractant. Quantities typically used at present, such as leading front distances, migrating cell numbers, etc., are unsatisfactory in this regard because their values are affected by many aspects of the assay system unrelated to cell behavioral properties. In this paper we demonstrate the measurement of cell migration parameters that do, in fact, characterize the intrinsic cell chemosensory movement responses using cell density profiles obtained in the linear under-agarose assay. These parameters are the random motility coefficient, mu, and the chemotaxis coefficient, chi, which appear in a theoretical expression for cell population migration. We propose a priori the dependence of chi on attractant concentration, based on an independent experimental correlation of individual cell orientation bias in an attractant gradient with a spatial difference in receptor occupancy. Our under-agarose population migration results are consistent with this proposition, allowing chemotaxis to be reliably characterized by a chemotactic sensitivity constant, chi 0, to which chi is directly proportional. Further, chi 0 has fundamental significance; it represents the reciprocal of the difference in number of bound receptors across cell dimensions required for directional orientation bias. In particular, for the system of human peripheral blood polymorphonuclear neutrophil leukocytes responding to FNLLP, we find that the chemotaxis coefficient is a function of attractant concentration, a following the expression: chi = chi 0NT0 f(a) S(a) Kd/(Kd + a)2 where Kd is the FNLLP-receptor equilibrium dissociation constant and NT0 is the total number of cell surface receptors for FNLLP. f(a) is the fraction of surface receptors remaining after down-regulation, and S(a) is the cell movement speed, both known functions of FNLLP concentration. We find that chi 0NT0 = 0.2 cm; according to a theoretical argument outlined in the Appendix this means that these cells exhibit 75% orientation toward higher attractant concentration when the absolute spatial difference in bound receptors is 0.0025NT0 over 10 micron. (For example, if NT0 = 50,000 this would correspond to a spatial difference of 125 bound receptors over 10 micron.) This result can be compared with estimates obtained from visual studies of individual neutrophils.     

Theoretical results for chemotactic response and drift of E. coli in a weak attractant gradient

The bacterium Escherichia coli (E. coli) moves in its natural environment in a series of straight runs, interrupted by tumbles which cause change of direction. It performs chemotaxis towards chemo-attractants by extending the duration of runs in the direction of the source. When there is a spatial gradient in the attractant concentration, this bias produces a drift velocity directed towards its source, whereas in a uniform concentration, E. coli adapts, almost perfectly in case of methyl aspartate. Recently, microfluidic experiments have measured the drift velocity of E. coli in precisely controlled attractant gradients, but no general theoretical expression for the same exists. With this motivation, we study an analytically soluble model here, based on the Barkai-Leibler model, originally introduced to explain the perfect adaptation. Rigorous mathematical expressions are obtained for the chemotactic response function and the drift velocity in the limit of weak gradients and under the assumption of completely random tumbles. The theoretical predictions compare favorably with experimental results, especially at high concentrations. We further show that the signal transduction network weakens the dependence of the drift on concentration, thus enhancing the range of sensitivity.     

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.     

Modeling cell gradient sensing and migration in competing chemoattractant fields

Directed cell migration mediates physiological and pathological processes. In particular, immune cell trafficking in tissues is crucial for inducing immune responses and is coordinated by multiple environmental cues such as chemoattractant gradients. Although the chemotaxis mechanism has been extensively studied, how cells integrate multiple chemotactic signals for effective trafficking and positioning in tissues is not clearly defined. Results from previous neutrophil chemotaxis experiments and modeling studies suggested that ligand-induced homologous receptor desensitization may provide an important mechanism for cell migration in competing chemoattractant gradients. However, the previous mathematical model is oversimplified to cell gradient sensing in one-dimensional (1-D) environment. To better understand the receptor desensitization mechanism for chemotactic navigation, we further developed the model to test the role of homologous receptor desensitization in regulating both cell gradient sensing and migration in different configurations of chemoattractant fields in two-dimension (2-D). Our results show that cells expressing normal desensitizable receptors preferentially orient and migrate toward the distant gradient in the presence of a second local competing gradient, which are consistent with the experimentally observed preferential migration of cells toward the distant attractant source and confirm the requirement of receptor desensitization for such migratory behaviors. Furthermore, our results are in qualitative agreement with the experimentally observed cell migration patterns in different configurations of competing chemoattractant fields.     

The chemotactic behavior of computer-based surrogate bacteria

BACKGROUND: Chemotaxis is the process by which organisms migrate toward nutrients and favorable environments and away from toxins and unfavorable environments. In many species of bacteria, this occurs when extracellular signals are detected by transmembrane receptors and relayed to flagellar motors, which control the cell's swimming behavior. RESULTS: We used a molecularly detailed reaction-kinetics model of the chemotaxis pathway in Escherichia coli coupled to a graphical display based on known swimming parameters to simulate the responses of bacteria to 2D gradients of attractants. The program gives the correct phenotype of over 60 mutants in which chemotaxis-pathway components are deleted or overexpressed and accurately reproduces the responses to pulses and step increases of attractant. In order to match the known sensitivity of bacteria to low concentrations of attractant, we had to introduce a set of "infectivity" reactions based on cooperative interactions between neighboring chemotaxis receptors in the membrane. In order to match the impulse response to a brief stimulus and to achieve an effective accumulation in a gradient, we also had to increase the activities of the adaptational enzymes CheR and CheB at least an order of magnitude greater than published values. Our simulations reveal that cells develop characteristic levels of receptor methylation and swimming behavior at different positions along a gradient. They also predict a distinctive "volcano" profile in some gradients, with peaks of cell density at intermediate concentrations of attractant. CONCLUSIONS: Our results display the potential use of computer-based bacteria as experimental objects for exploring subtleties of chemotactic behavior.     

Engineered single‐ and multi‐cell chemotaxis pathways in E. coli

We have engineered the chemotaxis system of Escherichia coli to respond to molecules that are not attractants for wild‐type cells. The system depends on an artificially introduced enzymatic activity that converts the target molecule into a ligand for an E. coli chemoreceptor, thereby enabling the cells to respond to the new attractant. Two systems were designed, and both showed robust chemotactic responses in semisolid and liquid media. The first incorporates an asparaginase enzyme and the native E. coli aspartate receptor to produce a response to asparagine; the second uses penicillin acylase and an engineered chemoreceptor for phenylacetic acid to produce a response to phenylacetyl glycine. In addition, by taking advantage of a ‘hitchhiker’ effect in which cells producing the ligand can induce chemotaxis of neighboring cells lacking enzymatic activity, we were able to design a more complex system that functions as a simple microbial consortium. The result effectively introduces a logical ‘AND’ into the system so that the population only swims towards the combined gradients of two attractants.     

Interactions between Caenorhabditis elegans individuals during chemotactic response

The chemotactic response of the nematode Caenorhabditis elegans is known to be affected by the population density on an assay plate, suggesting the existence of interactions between individual animals. To clarify the interactions between individuals during chemotaxis, we investigated the effect of population density at an attractant area on the chemotactic response to water-soluble sodium acetate and odorant diacetyl using wild-type N2 animals and daf-22 (m130) mutants, which have defective pheromone secretion but can sense pheromone. The chemotaxis index of N2 animals at 90 min of the assay negatively correlated with the number of animals on the assay plate regardless of the type of attractant used (p<0.01). On the other hand, there was no significant difference in the chemotaxis indices of daf-22 (m130) mutants for either of the attractants between the low-and high-population groups. When daf-22 (m130) mutants of a high population density were placed at the attractant location in advance, the chemotaxis index of N2 animals was almost the same as that in the control assay in which no animals were placed at the attractant location in advance. When N2 animals of a high population density were placed at the attractant location in advance, the chemotaxis indices of N2 animals and daf-22 (m130) mutants were significantly smaller than those obtained in the control assay (p<0.05). In the absence of an attractant, we observed a decline in the fraction of animals in the neighborhood of N2 animals of a high population density, although the nematodes were not influenced by daf-22 (m130) mutants of a high population density. These results suggest that the attraction of nematodes to chemicals is inhibited by an increase in the concentration of the pheromone generated by N2 animals at the attractant location.     

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.     

Analysis of chemotactic bacterial distributions in population migration assays using a mathematical model applicable to steep or shallow attractant gradients

The mathematical model developed by Riveroet al. (1989,Chem. Engng Sci. 44, 2881–2897) is applied to literature data measuring chemotactic bacterial population distributions in response to steep as well as shallow attractant gradients. This model is based on a fundamental picture of the sensing and response mechanisms of individual bacterial cells, and thus relates individual cell properties such as swimming speed and tumbling frequency to population parameters such as the random motility coefficient and the chemotactic sensitivity coefficient. Numerical solution of the model equations generates predicted bacterial density and attractant concentration profiles for any given experimental assay. We have previously validated the mathematical model from experimental work involving a step-change in the attractant gradient (Fordet al., 1991Biotechnol. Bioengng.37, 647–660; For and Lauffenburger, 1991,Biotechnol. Bioengng,37, 661–672). Within the context of this experimental assay, effects of attractant diffusion and consumption, random motility, and chemotactic sensitivity on the shape of the profiles are explored to enhance our understanding of this complex phenomenon. We have applied this model to various other types of gradients with successful intepretation of data reported by Dalquistet al. (1972,Nature New Biol. 236, 120–123) forSalmonella typhimurum validating the mathematical model and supportin the involvement of high and low affinity receptors for serine chemotaxis by these cells.     

The effect of amino acids on the motile behavior of Bacillus subtilis

Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays. The stimulation of velocity did not occur via the receptors for chemotaxis. Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant. After methionine starvation B. subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria. Addition of methionine to starved cells restored the response toward attractant. Revertants of B. subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant.Abbreviation O.D.₆₀₀ optical density measured at 600 nm