A differential role of extracellular signal-regulated kinase in stimulated PC12 pheochromocytoma cell movement

Rat pheochromocytoma PC12 cells have been widely used as a cell system for study of growth factor-stimulated cell functions. We report here that nerve growth factor (NGF) stimulated both chemotaxis (directional migration) and chemokinesis (random migration) of PC12 cells. Treatment with a MEK1/2-specific inhibitor (PD98059) or expression of a dominant negative variant of Ras differentially inhibited NGF-stimulated chemotaxis but not chemokinesis of PC12 cells. Priming of PC12 cells with NGF resulted in reduced extracellular signal-regulated kinase (ERK) activation and loss of chemotactic, but not chemokinetic, response. In addition, NGF stimulation of ERK is known to involve an early transient phase of activation followed by a late sustained phase of activation; in contrast, epidermal growth factor (EGF) elicits only early transient ERK activation. We observed that like NGF, EGF also stimulated both chemotaxis and chemokinesis, and treatment with PD98059 abolished the EGF-stimulated chemotaxis. Therefore, the early transient phase of ERK activation functioned in signaling chemotaxis; the late sustained phase of ERK activation did not seem to have an essential role. In addition, our results suggested that chemotactic signaling required a threshold level of ERK activation; at below threshold level of ERK activation, chemotaxis would not occur.     

The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses.

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.     

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.     

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.     

Elastin repeat peptides as chemoattractants for bovine aortic endothelial cells

Cultured bovine aortic endothelial cells migrate toward a concentration gradient of repeating elastin peptides, specifically the repeating nonamers Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro and Gly-Leu-Gly-Val-Gly-Ala-Gly-Val-Pro and the repeating hexamer Val-Gly-Val-Ala-Pro-Gly. Dose-response experiments demonstrate that the peak of activity occurs at 8 x 10(-8) M for the nonapeptides and 1 x 10(-8) M for the hexapeptide. Checkerboard assays establish that the movement is chemotaxis and not chemokinesis. Because of the concentration difference in the responsiveness between the nonapeptide and the hexapeptide, the cells can differentiate between the two types of repeats. The positive control for the chemotaxis studies was fibronectin.     

A simplified Boyden chamber assay for neutrophil chemotaxis based on quantitation of myeloperoxidase

Cell locomotion and chemotaxis are usually assayed by the Boyden chamber technique, in which the response is measured by microscopical counting of the cells migrated into a micropore filter. We report a simplified Boyden chamber method which utilizes myeloperoxidase (MPO) specific to neutrophilic and monocytic leukocytes. The chamber is incubated for a period long enough for the neutrophils to migrate through the first of two superimposed filters. The cells entering the second filter are then lysed and the released MPO activity is quantitated. Random migration, chemokinesis, and chemotaxis measurements of neutrophils were compared by the enzymatic and the conventional cell count methods. There was good agreement between the two methods (0.84 less than r less than 0.98). The intraassay precision of the enzymatic and the cell count methods was equal; the coefficients of variation were 14 and 15%, respectively. The enzymatic method provides a more objective, reliable, and rapid modification of the Boyden chamber assay for analysis of neutrophil chemotaxis.     

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.     

Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate

Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.     

Chemotaxis in Rhizobium meliloti strain L5.30

We have found that Rhizobium meliloti strain L5.30 exhibits positive chemotaxis towards some amino acids, sugars, exudates and extracts from roots of legume plants. From the investigated compounds sugars were better chemo-attractants than amino acids, but legume root substances were the best ones. Positive chemotaxis towards legume root compounds was supported by clouds of R. meliloti cells observed at the surface of alfalfa roots. A large deletion, in the nodABC region of the symbiotic megaplasmid (Sym), did not eliminate rhizobial chemotaxis.     

Chemotaxis by Naegleria fowleri for bacteria

Naegleria fowleri amebae demonstrated a chemotactic and chemokinetic response toward live cells and extracts of Escherichia coli and other bacterial species when experiments were performed using a blind-well chemotaxis chamber. The peptide N-formyl-methionyl-leucyl-phenylalanine acted as a chemokinetic rather than a chemotactic factor for N. fowleri amebae. Competition experiments in which nerve cell extracts or bacteria were placed on either side of the filter in chemotaxis chambers resulted in increased movement towards bacteria. A scanning electron microscopy study of the interaction of N. fowleri with different bacterial species confirmed that when the amebae were near ingestible bacteria they moved toward the bacteria by pseudopod formation. Naegleria fowleri appeared to respond to bacteria by three interrelated but distinct processes: chemokinesis, chemotaxis, and formation of food cups.     

Sensory transduction in the gliding bacterium Myxococcus xanthus

Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl-accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.     

Ideal and non-ideal concentration gradient propagation in chemotaxis studies

The shape and spatio-temporal development of a concentration gradient depend on the method and medium of its generation. Model gradients of low molecular weight (MW) dyes develop with predictable characteristics in 1% agarose. In contrast, any method of delivering dyes into free solution, e.g., via capillary pipets, produces anomalous gradients, unless the chambers are extremely shallow (less than 1 mm deep). Thus, most methods of chemo-attractant delivery which are popular in chemotaxis studies are incapable of propagating gradients by molecular diffusion; rather, they tend to be dominated by the effects of wholesale flow of the chemo-attractant and convection and turbulence of the free solution. This may have serious consequences for the interpretation of some chemotaxis experiments, since several gradient qualities of chemo-attractants (and probably morphogens) could influence cell behaviour. The possible effects on cells of the motion and development of gradients are also discussed.     

Investigate the role of PTEN in chemotaxis of human breast cancer cells

Chemotaxis plays an important role in metastasis of cancer cells. In the current study, we investigated the role of PTEN, a tumor suppressor, in chemotaxis of human breast cancer cells. Over-expression of PTEN inhibited EGF-induced chemotaxis, probably due to an overall reduction of PIP(3) levels. Disruption of PTEN by siRNA caused a marked decrease in chemokinesis, cell adhesion, and membrane spreading, resulting in a severe defect in chemotaxis. In PTEN disrupted cells, PDK1, AKT, and PKCzeta exhibited elevated basal activities, which prevented EGF-induced further activation of these molecules. In the absence of EGF, active PDK1 was detected on multiple directions of the plasma membranes of PTEN disrupted cells, which competed against EGF-induced gradient sensing. To confirm the biological relevance of in vitro studies, both PTEN disrupted cells and its parental human breast cancer cells were injected into tail veins of SCID mice. Mice injected with PTEN disrupted cancer cells showed a marked decrease in lung metastasis. Taken together, our data show that PTEN plays a non-redundant role in EGF-induced chemotaxis of human breast cancer cells, and an optimal level of PTEN is required in these responses.     

Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 cells by TGF-beta isoforms

Proliferation of mesenchymal precursors of osteogenic and chondrogenic cells and migration of these precursors to repair sites are important early steps in bone repair. Transforming growth factor-beta (TGF-beta) has been implicated in the promotion of bone repair and may have a role in these processes. Three isoforms of TGF-beta, TGF-beta1, -beta2, and -beta3, are expressed in fracture healing, however, their specific roles in the repair process are unknown. Differential actions of the TGF-beta isoforms on early events of bone repair were explored in the multipotent mesenchymal precursor cell line, C3H10T1/2. Cell migration was determined using a modified Boyden chamber in response to concentrations of each isoform ranging from 10(-12) to 10(-9) g/ml. All three isoforms demonstrated a dose-dependent chemotactic stimulation of untreated C3H10T1/2 cells. Checkerboard assays indicated that all three isoforms also stimulated chemokinesis of the untreated cells. C3H10T1/2 cells treated with all-trans-retinoic acid (ATRA) and expressing relatively higher levels of osteoblastic gene markers such as alkaline phosphatase and collagen type I, lower levels of chondrocytic gene markers collagen type II and aggrecan, and unchanged levels of the adipose marker adipsin did not demonstrate significant chemokinesis or chemotaxis in response to TGF-beta1 or -beta3 at concentrations ranging from 10(-12) to 10(-9) g/ml. In the ATRA-treated cells, TGF-beta2 stimulated a significant increase in chemotaxis only at the highest concentration tested. Cell proliferation was assessed by mitochondrial dehydrogenase activity and cell counts at TGF-beta concentrations from 10(-11) to 10(-8) g/ml. None of the TGF-beta isoforms stimulated cell proliferation in untreated or ATRA-treated C3H10T1/2 cells. Analysis of TGF-beta receptors (TGF-betaR1, -betaR2, and -betaR3) showed a 1.6- to 2.8-fold decrease in mRNA expression of these receptors in ATRA-treated cells. In conclusion: (1) while all three TGF-beta isoforms stimulate chemotaxis/chemokinesis of multipotent C3H10T1/2 cells, TGF-beta1 and -beta3 do not stimulate chemotaxis in C3H10T1/2 cells treated with ATRA while TGF-beta2 stimulated chemotaxis only at the highest concentration tested. (2) TGF-beta isoforms do not appear to stimulate cell proliferation in C3H10T1/2 cells in either a multipotent state or after ATRA treatment when expressing higher levels of alkaline phosphatase and collagen type I gene markers. (3) Decrease in mRNA expression for TGF-betaR1, -betaR2, and -betaR3 upon ATRA treatment could potentially explain the lack of chemotaxis/chemokinesis in these cells expressing higher levels of alkaline phosphatase and collagen type I.     

A 'chemotactic dipole' mechanism for large-scale vortex motion during primitive streak formation in the chick embryo

Primitive streak formation in the chick embryo involves significant coordinated cell movement lateral to the streak, in addition to the posterior-anterior movement of cells in the streak proper. Cells lateral to the streak are observed to undergo 'polonaise movements', i.e. two large counter-rotating vortices, reminiscent of eddies in a fluid. In this paper, we propose a mechanism for these movement patterns which relies on chemotactic signals emitted by a dipolar configuration of cells in the posterior region of the epiblast. The 'chemotactic dipole' consists of adjacent regions of cells emitting chemo-attractants and chemo-repellents. We motivate this idea using a mathematical analogy between chemotaxis and electrostatics, and test this idea using large-scale computer simulations. We implement active cell response to both neighboring mechanical interactions and chemotactic gradients using the Subcellular Element Model. Simulations show the emergence of large-scale vortices of cell movement. The length and time scales of vortex formation are in reasonable agreement with experimental data. We also provide quantitative estimates for the robustness of the chemotaxis dipole mechanism, which indicate that the mechanism has an error tolerance of about 10% to variation in chemotactic parameters, assuming that only 1% of the cell population is involved in emitting signals. This tolerance increases for larger populations of cells emitting signals.     

Thrombin-induced chemotaxis and aggregation of neutrophils

Thrombin-induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (10(8) cells/ml) exhibited maximum chemotactic migration towards 10(-8)M human alpha-thrombin, 10(-8)M gamma-thrombin (which lacks the fibrinogen site), and 10(-12)MD-Phe-Pro-Arg-CH2-alpha-thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (10(8) cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10(-11)M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, alpha-thrombin, and gamma-thrombin. The responses of both human and sheep neutrophils to D-Phe-Pro-Arg-CH2-alpha-thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin-induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.     

Solution structure of a tmRNA-binding protein,SmpB, from Thermus thermophilus

The molecular mechanisms that govern cell movement are the subject of intense study, as they impact biologically and medically important processes such as leukocyte chemotaxis and angiogenesis, among others. We demonstrate that leukocyte chemotaxis is prevented by the macrolide immunosuppressant rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR)/ribosomal p70-S6 kinase (p70S6K) pathway. Both neutrophil chemotaxis and chemokinesis elicited by granulocyte-macrophage colony-stimulating factor (GM-CSF) were strongly inhibited by rapamycin with an IC₅₀ of 0.3 nM. Inhibition, although at a higher dose, was also observed when the chemoattractant was interleukin-8. As for the mechanism, rapamycin targeted the increase of phosphorylation of p70S6K due to GM-CSF treatment, as demonstrated with specific anti-p70S6K immunoprecipitation and subsequent immunoblotting with anti-T⁴²¹/S⁴²⁴ antibodies. Rapamycin also inhibited GM-CSF-induced actin polymerization, a hallmark of leukocyte migration. The specificity of the effect of rapamycin was confirmed by the use of the structural analog FK506, which did not have a significant effect on chemotaxis but effectively rescued rapamycin-induced p70S6K inhibition. This was expected from a competitive effect of both molecules on FK506-binding proteins (FKBP). Additionally, GM-CSF-induced chemotaxis was completely (>90%) blocked by a combination of rapamycin and the MAPK kinase (MEK) inhibitor PD-98059. In summary, the results presented here indicate for the first time that rapamycin, at sub-nanomolar concentrations, inhibits GM-CSF-induced chemotaxis and chemokinesis. This serves to underscore the relevance of the mTOR/S6K pathway in neutrophil migration.     

The in vitro effects of histamine and metiamide on neutrophil motility and their relationship to intracellular cyclic nucleotide levels.

Histamine at concentrations of 1 x 10(-5) M to 5 x 10(-5) M consistently increased neutrophil movement as measured in Boyden chambers. This effect was entirely caused by stimulation of chemokinesis (stimulated random migration) and true chemotaxis was inhibited by these concentrations. This inhibition of chemotaxis could be abolished by pretreatment with metiamide, an H-2 receptor antagonist, and levamisole, but not by diphenylhydramine, an H-1 receptor antagonist. Metiamide at similar concentrations produced a mild stimulation of chemokinesis but has no effect on true chemotaxis. The histamine effects on neutrophil motility were associated with increased levels of intracellular cAMP wehreas cAMP levels were unaffected. Agents known to elevate intracellular cAMP levels produced effects on neutrophil motility similar to those of histamine. It is suggested that histamine exerts a 2-fold effect on neutrophil motility mediated via an H-2 receptor site and associated with elevated levels of cAMP.     

Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells

AMP-activated protein kinase (AMPK) is a key energy sensor, known to regulate energy metabolism in diverse cell types. Hypoxia is encountered frequently in the microenvironments of inflammatory lesions and is a critical regulator of function in inflammatory cells. Energy deficiency is one of the consequences of hypoxia, but its potential role in modulating leucocyte function has received little attention. Using micropore chemotaxis assays to assess migratory responses of the monocyte-like cell line U937, it was found that the AMPK activators AICAR and phenformin rapidly reduced random migration (chemokinesis) as well as chemotaxis due to stromal cell-derived factor (SDF)1alpha. There was an approximate 50% reduction in both chemokinesis and chemotaxis following 30 min preincubation with both AICAR and phenformin (P < 0.01), and this continued with up to 24 h preincubation. The binding of SDF1alpha to its receptor CXCR4 was unaltered, suggesting AMPK was acting on downstream intracellular signalling pathways important in cell migration. As AMPK and statins are known to inhibit HMG CoA reductase, and both reduce cell migration, the effect of mevastatin on U937 cells was compared with AMPK activators. Mevastatin inhibited cell migration but required 24 h preincubation. As expected, the inhibitory effect of mevastatin was associated with altered subcellular localization of the Rho GTPases, RhoA and cdc42, indicating decreased prenylation of these molecules. Although the effect of AMPK activation was partially reversed by mevalonate, this was not associated with altered subcellular localization of Rho GTPases. The data suggest that activation of AMPK has a general effect on cell movement in U937 cells, and this is not due to inhibition of HMG CoA reductase. These are the first data to show an effect of AMPK on cell movement, and suggest a fundamental role for energy deficiency in regulating cellular behaviour.