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Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coH RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.

[Na.sub.v]BP, found in alkaliphilic Bacillus pseudofirmus OF4, is a member of the bacterial voltage-gated Na+ channel superfamily. The alkaliphile requires [Na.sub.v]BP for normal chemotaxis responses and for optimal pH homeostasis during a shift to alkaline conditions at suboptimally low [Na.sup.+] concentrations. We hypothesized that interaction of [Na.sub.v]BP with one or more other proteins in vivo, specifically methyl-accepting chemotaxis proteins (MCPs), is involved in activation of the channel under the pH conditions that exist in the extremophile and could underpin its role in chemotaxis; MCPs transduce chemotactic signals and generally localize to cell poles of rod-shaped cells. Here, immunofluorescence microscopy and fluorescent protein fusion studies showed that an alkaliphile protein (designated McpX) that cross-reacts with antibodies raised against Bacillus subtilis McpB co-localizes with [Na.sub.v]BP at the cell poles of B. pseudofirmus OF4. In a mutant in which [Na.sub.v]BP-encoding ncbA is deleted, the content of McpX was close to the wild-type level but McpX was significantly delocalized. A mutant of B. pseudofirmus OF4 was constructed in which cheAW expression was disrupted to assess whether this mutation impaired polar localization of McpX, as expected from studies in Escherichia coli and Salmonella, and, if so, whether [Na.sub.v]BP would be similarly affected. Polar localization of both McpX and [Na.sub.v]BP was decreased in the cheAW mutant. The results suggest interactions between McpX and [Na.sub.v]BP that affect their co-localization. The inverse chemotaxis phenotype of ncbA mutants may result in part from MCP delocalization.

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration. muscle repair; macrophage; chemotaxis

During development of the nervous system, the tip of a growing axon, the growth cone (GC), must respond accurately to stimuli that direct its growth. This axonal navigation depends on extracellular concentration gradients of numerous guidance cues, including GABA. GCs can detect even weak directional signals, yet the mechanisms underlying this sensitivity remain unclear. Past studies in other eukaryotic chemotactic systems have pointed to the role of the spatial reorganization of the transduction pathway in their sensitive response. Here we have developed a single-molecule assay to observe individual [GABA.sub.A] receptors ([GABA.sub.A]Rs) in the plasma membrane of nerve GCs subjected to directional stimuli. We report that in the presence of an external GABA gradient [GABA.sub.A]Rs redistribute asymmetrically across the GC toward the gradient source. Single-particle tracking of [GABA.sub.A]RS shows that the redistribution results from transient interactions between the receptors and the microtubules. Moreover, the relocalization is accompanied by an enhancement in the asymmetry of intracellular calcium concentration. Altogether, our results reveal a microtubule-dependent polarized reorganization of chemoreceptors at the cell surface and suggest that this polarization serves as an amplification step in GABA gradient sensing by nerve GCs. axonal guidance | chemotaxis | single molecule | polarity

Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the [beta]5-[alpha]2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7. doi:10.1128/JB.01098-06

Mast cell recruitment is implicated in many physiological functions and several diseases. It depends on microenvironmental factors, including hormones. We have investigated the effect of progesterone on the migration of HMC-[1.sup.560] mast cells toward CXCL12, a chemokine that controls the migration of mast cells into tissues. HMC-[1.sup.560] mast cells were incubated with 1 nM to 1 [micro]M progesterone for 24 h. Controls were run without progesterone. Cell migration toward CXCL12 was monitored with an in vitro assay, and statistical analysis of repeated experiments revealed that progesterone significantly reduced cell migration without increasing the number of apoptotic cells (P = 0.0084, n = 7). Differences between progesterone-treated and untreated cells were significant at 1 [micro]M (P < 0.01, n = 7). Cells incubated with 1 [micro]M progesterone showed no rearrangment of actin filaments in response to CXCL12. Progesterone also reduced the calcium response to CXCL12 and Akt phosphorylation. Cells incubated with progesterone had one-half the control concentrations of CXCR4 (mRNA, total protein, and membrane-bound protein). Progesterone also inhibited the migration of HMC-[1.sup.560] cells transfected with hPR-B-pSG5 plasmid, which contained 2.5 times as much PR-B as the control. These transfected cells responded differently (P < 0.05, n = 5) from untreated cells to 1 nM progesterone. We conclude that progesterone reduces mast cell migration toward CXCL12 and that CXCR4 may be a progesterone target in mast cells. chemotaxis; sex steroid; immune system doi:10.1152/ajpendo.00286.2006

Signal transduction in bacterial chemotaxis is initiated by the binding of extracellular ligands to a specialized family of methyl-accepting chemoreceptor proteins. Chemoreceptors cluster at distinct regions of the cell and form stable ternary complexes with the histidine autokinase CheA and the adapter protein CheW. Here we report the direct visualization and spatial organization of chemoreceptor arrays in intact Escherichia coli cells by using cryo-electron tomography and biochemical techniques. In wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing nonfunctional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be reestablished by increasing cellular levels of CheA and CheW. Our results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW. cryo-tomography | signal transduction | molecular architecture

As an important model for transmembrane signaling, methylaccepting chemotaxis proteins (MCPs) have been extensively studied by using genetic, biochemical, and structural techniques. However, details of the molecular mechanism of signaling are still not well understood. The availability of genomic information for hundreds of species enables the identification of features in protein sequences that are conserved over long evolutionary distances and thus are critically important for function. We carried out a large-scale comparative genomic analysis of the MCP signaling and adaptation domain family and identified features that appear to be critical for receptor structure and function. Based on domain length and sequence conservation, we identified seven major MCP classes and three distinct structural regions within the cytoplasmic domain: signaling, methylation, and flexible bundle subdomains. The flexible bundle subdomain, not previously recognized in MCPs, is a conserved element that appears to be important for signal transduction. Remarkably, the N- and C-terminal helical arms of the cytoplasmic domain maintain symmetry in length and register despite dramatic variation, from 24 to 64 7-aa heptads in overall domain length. Loss of symmetry is observed in some MCPs, where it is concomitant with specific changes in the sensory module. Each major MCP class has a distinct pattern of predicted methylation sites that is well supported by experimental data. Our findings indicate that signaling and adaptation functions within the MCP cytoplasmic domain are tightly coupled, and that their coevolution has contributed to the significant diversity in chemotaxis mechanisms among different organisms. chemotaxis | methyl-accepting chemotaxis protein | signal transduction

We demonstrate the use of Escherichia coli and their chemotactic characteristics to enhance mixing in a microchannel in a controlled and bi-directional manner. The presence of a chemoattractant in one arm of a three-junction microchannel results in an asymmetric increase in the effective diffusion coefficient of extremely high molecular weight TMR-Dextran (MW 2 000 000), which rises linearly with the concentration of attractant from a baseline value of 8-42 [micro][m.sup.2]/s at a concentration of 0.1 M. The response to a repellent is similar, with the opposite bias.

Host-seeking behavior by parasitic nematodes relies heavily on chemical cues emanating from potential hosts. Nonspecific cues for Strongyloides stercoralis, a nematode that infects humans and a few other mammals, include carbon dioxide and sodium chloride; however, the characteristic species specificity of this parasite suggested the existence of other, more specific cues. Here we show that the infective larva of S. stercoralis is strongly attracted to an extract of mammalian skin and that the active component in this skin extract is urocanic acid. Urocanic acid, a histidine metabolite, is particularly abundant in mammalian skin and skin secretions, suggesting that it serves as an attractant specific to mammalian hosts. The attractant activity of urocanic acid is suppressed by divalent metal ions, suggesting a possible strategy for preventing infection. histidine | host-seeking | chemotaxis | topical preventative

Adenosine is produced during cellular hypoxia and apoptosis, resulting in elevated tissue levels at sites of injury. Adenosine is also known to regulate a number of cellular responses to injury, but its role in hepatic stellate cell (HSC) biology and liver fibrosis is poorly understood. We tested the effect of adenosine on the cytosolic [Ca.sup.2+] concentration, chemotaxis, and upregulation of activation markers in HSCs. We showed that adenosine did not induce an increase in the cytosolic [Ca.sup.2+] concentration in LX-2 cells and, in addition, inhibited increases in the cytosolic [Ca.sup.2+] concentration in response to ATP and PDGF. Using a Transwell system, we showed that adenosine strongly inhibited PDGF-induced HSC chemotaxis in a dose-dependent manner. This inhibition was mediated via the [A.sub.2]a receptor, was reversible, was reproduced by forskolin, and was blocked by the adenylate cyclase inhibitor 2,5-dideoxyadenosine. Adenosine also upregulated the production of TGF-[beta] and collagen I mRNA. In conclusion, adenosine reversibly inhibits [Ca.sup.2+] fluxes and chemotaxis of HSCs and upregulates TGF-[beta] and collagen I mRNA. We propose that adenosine provides 1) a 'stop' signal to HSCs when they reach sites of tissue injury with high adenosine concentrations and 2) stimulates transdifferentiation of HSCs by upregulating collagen and TGF-[beta] production. platelet-derived growth factor; [Ca.sup.2+]; fibrosis

Chemotaxis of bacteria requires regulated methylation of chemo-receptors. However, despite considerable effort in the 1980s, transmethylation has never been established as a component of eukaryotic cell chemotaxis. S-adenosylhomocysteine (SAH), the product formed when the methyl group of the universal donor S-adenosylmethionine (SAM) is transferred to an acceptor molecule, is a potent inhibitor of all transmethylation reactions. In eukaryotic cells, this inhibition is relieved by hydrolysis of SAH to adenosine and homocysteine catalyzed by SAH hydrolase (SAHH). We now report that SAHH, which is diffuse in the cytoplasm of nonmotile Dictyostelium amoebae and human neutrophils, concentrates with F-actin in pseudopods at the front of motile, chemotaxing cells, but is not present in filopodia or at the very leading edge. Tubercidin, an inhibitor of SAHH, inhibits both chemotaxis and chemotaxis-dependent cell streaming of Dictyostelium, and chemotaxis of neutrophils at concentrations that have little effect on cell viability. Tubercidin does not inhibit starvation-induced expression of the cAMP receptor, cAR1, or G protein-mediated stimulation of adenylyl cyclase activity and actin polymerization in Dictyostelium. Tubercidin has no effect on either capping of Con A receptors or phagocytosis in Dictyostelium. These results add SAHH to the list of proteins that redistribute in response to chemotactic signals in Dictyostelium and neutrophils and strongly suggest a role for transmethylation in chemotaxis of eukaryotic cells. chemotaxis | Dictyostelium | neutrophils | tubercidin

The dimorphic prosthecate bacteria (DPB) are [alpha]-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

Escherichia coli chemotaxis is mediated by membrane receptor/ histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, [IzTar.sub.c], that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from [IzTar.sub.c]. Thus, the organization of these receptor/signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo. CheA | histidine kinase | serine receptor | signal transduction

The chemotaxis network in Escherichia coli is remarkable for its sensitivity to small relative changes in the concentrations of multiple chemical signals over a broad range of ambient concentrations. Key to this sensitivity is an adaptation system that relies on methylation and demethylation (or deamidation) of specific modification sites of the chemoreceptors by the enzymes CheR and CheB, respectively. It was recently discovered that these enzymes can access five to seven receptors when tethered to a particular receptor. We show that these 'assistance neighborhoods' are necessary for precise adaptation in a model for signaling by clusters of chemoreceptors. In agreement with experiment, model clusters composed of receptors of different types exhibit high sensitivity and precise adaptation over a wide range of chemical concentrations and the response of adapted clusters to addition/ removal of attractant scales with free-energy change. We predict two limits of precise adaptation at large attractant concentrations: Either receptors reach full methylation and turn off, or receptors become saturated and cease to respond to attractant but retain their adapted activity. Monod-Wyman-Changeux model | receptor clustering | two-state receptors | signaling

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate ([PIP.sub.3]) at the leading edge. On the other hand, we found that DOCK2 associates with [PIP.sub.3] and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through [PIP.sub.3]-dependent membrane translocation and Rac activation.

Many eukaryotic cells, including Dictyostelium discoideum amoebae, fibroblasts, and neutrophils, are able to respond to chemoattractant gradients with high sensitivity. Recent studies have demonstrated that, after the introduction of a chemoattractant gradient, several chemotaxis pathway components exhibit a subcellular reorganization that cannot be described as a simple amplification of the external gradient, instead, this reorganization has the characteristics of a switch, leading to a well defined front and back. Here, we propose a directional sensing mechanism in which two second messengers are produced at equal rates. The diffusion of one of them, coupled with an inactivation scheme, ensures a switch-like response to external gradients for a large range of gradient steepness and average concentration. Furthermore, our model is able to reverse the subcellular organization rapidly, and its response to multiple simultaneous chemoattractant sources is in good agreement with recent experimental results. Finally, we propose that the dynamics of a heterotrimeric G protein might allow for a specific biochemical realization of our model. dynamics | modeling

Motile bacteria follow gradients of attractant and repellent chemicals with high sensitivity. Their chemoreceptors are physically clustered, which may enable them to function as a cooperative array. Although native chemoreceptor molecules are typically transmembrane homodimers, they appear to associate through their cytoplasmic tips to form trimers of dimers, which may be an important architectural element in the assembly and operation of receptor clusters. The five receptors of Escherichia coli that mediate most of its chemotactic and aerotactic behaviors have identical trimer contact residues and have been shown by in vivo crosslinking methods to form mixed trimers of dimers. Mutations at the trimer contact sites of Tsr, the serine chemoreceptor, invariably abrogate Tsr function, but some of those lesions (designated Tsr*) are epistatic and block the function of heterologous chemoreceptors. We isolated and characterized mutations (designated Tar^) in the aspartate chemoreceptor that restored function to Tsr* receptors. The suppressors arose at or near the Tar trimer contact sites and acted in an allele-specific fashion on Tsr* partners. Alone, many Tar^ receptors were unable to mediate chemotactic responses to aspartate, but all formed clusters with varying efficiencies. Most of those Tar^ receptors were epistatic to WT Tsr, but some regained Tar function in combination with a suppressible Tsr* partner. Tar^-Tsr* suppression most likely occurs through compensatory changes in the conformation or dynamics of a mixed receptor signaling complex, presumably based on trimer-of-dimer interactions. These collaborative teams may be responsible for the high-gain signaling properties of bacterial chemoreceptors. chemotaxis | epistasis | receptor clustering | signaling teams | trimers of dimers

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis and ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells and provide access to the systemic circulation. In addition to well established growth factors and inflammatory mediators that promote capillary sprouting and endothelial cell growth and migration, an emerging body of evidence supports a previously unrecognized function for axon guidance molecules in regulation of blood vessel development. Here we show that semaphorin 4D (Sema4D), a protein originally shown to regulate axonal growth cone guidance in the developing central nervous system through its receptor, plexin-B1, is highly expressed in cell lines derived from head and neck squamous cell carcinomas (HNSCCs) at both the protein and message level. Immunohistochemical analysis of a large collection of HNSCC specimens revealed high levels of Sema4D in a cell surface pattern in invading islands of transformed epithelial cells, but not in normal and noninvasive dysplastic epithelium. A similar pattern was observed in malignant cells from prostate, colon, breast, and lung cancer tissues. When shed from HNSCC cells, Sema4D stimulates endothelial cell migration, which can be prevented by Sema4D-blocking antibodies and by Sema4D knockdown. Furthermore, knocking down Sema4D by lentiviral expression of Sema4D shRNA reduces dramatically the size and vascularity of HNSCC tumor xenografts. These findings indicate that expression of Sema4D is a frequently used strategy by which a wide variety of carcinomas may promote angiogenesis, and therefore is a possible therapeutic target for the treatment of these malignancies. head and neck cancer | c-Met protein | plexin | chemotaxis | endothelium

Cell division is a carefully orchestrated procedure. Bacterial cells have intricate mechanisms to ensure that genetic material is copied, proofread, and accurately partitioned into daughter cells. Partitioning now appears to also occur for some cytoplasmic proteins. Previously, using chromosomal fluorescent protein fusions, we demonstrated that a subset of Rhodobacter sphaeroides chemotaxis proteins colocalize to a discrete region within the bacterial cytoplasm. Using TIpT-yellow fluorescent protein as a marker for the position of the cytoplasmic protein clusters, we show most cells contain either one cluster localized at mid-cell or two clusters at the one-fourth and three-fourths positions of cell length. The number and positioning of these protein clusters depend on a previously unrecognized bacterial protein positioning factor, PpfA, which has homology to bacterial type I DNA partitioning factors. These data suggest that there is a mechanism involved in partitioning some cytoplasmic proteins upon cell division that is analogous to a mechanism seen for plasmid and chromosomal DNA. chemoreceptor | chemotaxis | partition | Rhodobacter

Sperm chemotaxis is an area of significant interest to scientists involved in reproductive science. Understanding how and when sperm cells are attracted to the egg could have profound effects on reproduction and contraception. In an effort to systematically study this problem, we have fabricated and evaluated a microfluidic device to measure sperm chemotaxis. The device was designed with a flow-through configuration using a spatially and temporally stable chemical gradient. Mouse sperm cells were introduced into the chemotaxis chamber between confluent flows of mouse ovary extract and buffer. The sperm experiencing chemotaxis swam toward the extract and were counted relative to those that swam toward the buffer. The ovary extracts were diluted from 102 to 107 times, and each extract dilution was screened for chemotaxis. Four out of six ovaries showed a strong chemotactic response at extract dilutions of [10.sup.-3] to [10.sup.-5]. This device provided a convenient, disposable platform on which to conduct chemotaxis assays, and the flow-through design overcomes difficulties associated with distinguishing chemotaxis from trapping.

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/ VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis.

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.

We looked for a feedback system in Escherichia coil that might sense the rotational bias of flagellar motors and regulate the activity of the chemotaxis receptor kinase. Our search was based on the assumption that any machinery that senses rotational bias will be perturbed if flagellar rotation stops. We monitored the activity of the kinase in swimming cells by bioluminescence resonance energy transfer (BRET) between Renilla luciferase fused to the phosphatase, CheZ, and yellow fluorescent protein fused to the response regulator, CheY. Then we jammed the flagellar motors by adding an antifilament antibody that crosslinks adjacent filaments in flagellar bundles. At steady state, the rate of phosphorylation of CheY is equal to the rate of dephosphorylation of CheY-P, which is proportional to the degree of association between CheZ and CheY-P, the quantity sensed by BRET. No changes were observed, even upon addition of an amount of antibody that stopped the swimming of >95% of cells within a few seconds. So, the kinase does not appear to be sensitive to motor output. The BRET technique is complementary to one based on FRET, described previously. Its reliability was confirmed by measurements of the response of cells to the addition of attractants. Escherichia coli | signal transduction | Renilla | luciferase

The chemotaxis network in Escherichia coil is remarkable for its sensitivity to small relative changes in the concentrations of multiple chemical signals. We present a model for signal integration by mixed clusters of interacting two-state chemoreceptors. Our model results compare favorably to the results obtained by Sourjik and Berg with in vivo fluorescence resonance energy transfer. Importantly, we identify two distinct regimes of behavior, depending on the relative energies of the two states of the receptors. In regime I, coupling of receptors leads to high sensitivity, while in regime II, coupling of receptors leads to high cooperativity, i.e., high Hill coefficient. For homogeneous receptors, we predict an observable transition between regime I and regime II with increasing receptor methylation or amidation. chemotaxis | Monod, Wyman, and Changeux model | receptor clustering

The heterotrimeric G protein [G.sub.o] is abundantly expressed in the mammalian nervous system and modulates neural activities in response to various ligands. However, [G.sub.o]'s functions in living animals are less well understood. Here, we demonstrate that GOA-1 [G.sub.o][alpha] has a fundamental role in olfactory adaptation in Caenorhabditis elegans. Impairment of GOA-1 [G.sub.o][alpha] function and excessive activation of EGL-30 [G.sub.o][alpha] cause a defect in adaptation to AWC-sensed odorants. These pathways antagonistically modulate olfactory adaptation in AWC chemosensory neurons. Wild-type animals treated with phorbol esters and double-mutant animals of diacylglycerol (DAG) kinases, dgk-3; dgk-1, also have a defect in adaptation, suggesting that elevated DAG signals disrupt normal adaptation. Constitutively active GOA-1 can suppress the adaptation defect of dgk-3; dgk-1 double mutants, whereas it fails to suppress the adaptation defect of animals with constitutively active EGL-30, implying that GOA-1 acts upstream of EGL-30 in olfactory adaptation. Our results suggest that down-regulation of EGL-30-DAG signaling by GOA-1 underlies olfactory adaptation and plasticity of chemotaxis. chemotaxis | G protein

Various confocal microscopic techniques and FACScan are used to report that the members of the cohort containing beta-chemokines have much higher numbers of lower buoyant-density neutrophiles in their blood. The results show that the innate immunity of the cohort plays a very significant role in inhibiting the transmission of HIV, as the CC chemokines induce neutrophiles to chemotaxin degranulation and alpha-release of the cells.

The assembly and function of the mechanism of chemotaxis in Escherichia coli is discussed. Multimeric protein assemblies control the level of phosphorylation of a response regulator, CheY.

Leukotriene [B.sub.4] (LT[B.sub.4]), a potent leukocyte chemoattractant derived from the 5-lipoxygenase metabolism of arachidonic acid, exerts its action by means of specific cell surface receptors, denoted BL[T.sub.1] and BL[T.sub.2]. In this study, BL[T.sub.1] receptor proteins were detected in human carotid artery atherosclerotic plaques, colocalizing with markers for macrophages, endothelial cells, and vascular smooth muscle cells (SMC). Challenge of human coronary artery SMC with either LT[B.sub.4] or U75302, a partial agonist that is selective for the BL[T.sub.1] receptor, induced an [approximately equal to] 4-fold increase of whole-cell currents by using the patch--clamp technique, indicating that these cells express functional BL[T.sub.1] receptors. LT[B.sub.4] induced migration and proliferation of SMC in vitro, and treatment with the BLT receptor antagonist BIlL 284 (10 mg/kg, once daily) for 14 days after carotid artery balloon injury in vivo inhibited intimal hyperplasia in rats. In the latter model, SMC derived from the intima exhibited increased levels of BL[T.sub.1] receptor mRNA compared with medial SMC. BLT receptor up-regulation in the intima in vivo, as well as that induced by IL-1[beta] in vitro, were prevented by transfection with a dominant-negative form of I[kappa] kinase [beta] carried by adenovirus, indicating that BL[T.sub.1] receptor expression depends on NF-[kappa]B. These results show that LT[B.sub.4] activates functional BL[T.sub.1] receptors on vascular SMC, inducing chemotaxis and proliferation, and that BL[T.sub.1] receptors were up-regulated through an l[kappa] kinase [beta]/NF-[kappa]B-dependent pathway. Inhibition of LT[B.sub.4]/BL[T.sub.1] signaling during the response to vascular injury reduced intimal hyperplasia, suggesting this pathway as a possible target for therapy. restenosis | cell proliferation | chemotaxis | lipoxygenase | eicosanoids

A soluble methyl-accepting chemotaxis protein (MCP) of Pseudomonas aeruginosa, McpS, showed polar localization by immunofluorescence microscopy. Overexpression of McpS resulted in a dominant-negative effect on chemotaxis and caused a loss of polar clustering of the general MCP population. The polar localization of a soluble MCP defines a third, and unexpected, paradigm for cellular MCP localization.

The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers that bind the cytoplasmic proteins CheA and CheW to form ternary signaling complexes clustered at the cell poles. We used a trifunctional crosslinking reagent targeted to cysteine residues in the aspartate (Tar) and serine (Tsr) receptors to obtain in vivo snapshots of trimer composition in the receptor population. To analyze the dynamics of trimer formation, we followed the appearance of mixed trimers when cells expressing Tar were induced for the expression of Tsr and treated with the crosslinker shortly after the onset of induction. In the absence of CheA or CheW, preformed Tar trimers exchanged partners readily with newly made Tsr. Conversely, in the presence of CheA and CheW, receptor trimers seldom exchanged partners, irrespective of the presence or absence of attractants. The C-terminal receptor-coupling domain of the CheA kinase, which contains binding determinants for the CheW protein, was essential for conferring low exchangeability to the preformed trimers of dimers. CheW also was required for this effect, but, unlike CheA, overexpression of CheW interfered with trimer formation and chemotactic behavior. The CheW effect probably occurs through binding interactions that mask the receptor sites needed for trimer formation. We propose that clustered receptors are organized in mixed trimers of dimers through binding interactions with CheA and CheW, which play distinctly different architectural roles. Moreover, once complete signaling teams have formed, they no longer undergo dynamic exchange of receptor members. chemotaxis | epistasis | receptor clustering | signaling teams | trimer of dimers

Cell migration plays important roles in embryonic development and inflammation, and this process is highly regulated to ensure tissue homeostasis. A number of barriers exist to prevent the inappropriate migration of leukocytes into healthy peripheral tissues, including retention of these cells in the inactive state and maintenance of the integrity and charge of the vascular endothelium. However, active signals also are likely to exist that can repulse cells or abolish existing cell migration. One such paradigm exists in the developing nervous system, where neuronal migration is mediated by a balance between chemoattractive and chemorepulsive signals. The ability of the guidance molecule netrin-1 to repulse or abolish attraction of neuronal cells expressing the UNC5b receptor makes it an attractive candidate for the regulation of inflammatory cell migration. Here, we show that netrin-1 is expressed on vascular endothelium, where it is regulated by infection and inflammatory cytokines. The netrin-1 receptor UNC5b is strongly expressed by leukocytes, upon which netrin-1 acts as a potent inhibitor of migration to different chemotactic stimuli both in vivo and in vitro. These data suggest that endothelial expression of netrin-1 may inhibit basal cell migration into tissues and that its down-regulation with the onset of sepsis/inflammation may facilitate leukocyte recruitment. chemotaxis | inflammation | guidance cue

Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking the angiogenesis inhibitor thrombospondin-1 (TSP1) exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several proangiogenic responses of endothelial cells to NO. NO has a biphasic effect on endothelial cell proliferation. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner. TSP1 potently inhibits this response both upstream and downstream of cGMP. NO-stimulated endothelial cell responses are inhibited by recombinant type 1 repeats of TSP1 and a CD36 agonist antibody but not by the N-terminal portion of TSP1, suggesting that CD36 or a related receptor mediates these effects. These results demonstrate a potent antagonism between TSP1 and proangiogenic signaling downstream of NO. Further elucidation of this inhibitory signaling pathway may identify new molecular targets to regulate pathological angiogenesis. angiogenesis | cell adhesion | chemotaxis | proliferation | CD36

Many crucial biological processes operate with surprisingly small numbers of molecules, and there is renewed interest in analyzing the impact of noise associated with these small numbers. Twenty-five years ago, Berg and Purcell showed that bacterial chemotaxis, where a single-celled organism must respond to small changes in concentration of chemicals outside the cell, is limited directly by molecule counting noise and that aspects of the bacteria's behavioral and computational strategies must be chosen to minimize the effects of this noise. Here, we revisit and generalize their arguments to estimate the physical limits to signaling processes within the cell and argue that recent experiments are consistent with performance approaching these limits.

Byline: T. A. Rupprecht (1), U. Koedel (1), B. Muhlberger (2), B. Wilske (3), A. Fontana (4), H.-W. Pfister (1) Keywords: CXCL11; I--TAC; neuroborreliosis; chemotaxis; chemokines; Boyden chamber Abstract: We investigated the role of the CXCR3 chemokine CXCL11 (I--TAC) for leukocyte recruitment to the CSF in neuroborreliosis (NB). CXCL11 levels in the CSF of 17 patients with acute NB were elevated compared with 20 non--inflammatory controls (100.1 vs. 54.1pg/ml, p = 0.002). Using a modified Boyden chamber chemotaxis--assay, the CSF of patients with acute NB was more chemotactic than the control CSF--samples (p = 0.001). A strong correlation (p &lt 0.001) between CXCL11 levels, chemotactic activity and CSF--white cell count was detected. Though the chemotactic activity of CSF from NB patients was only partially reduced by a human recombinant CXCL11 antibody, these findings suggest that, among other chemotactic factors, CXCL11 may play a role in the chemotaxis of mononuclear cells in NB. Author Affiliation: (1) Dept. of Neurology, Klinikum Grosshadern Ludwig--Maximilians University, Marchioninistr. 15, 81377, Munich, Germany (2) Dept. of Neurology, University of Cologne, Koln, Germany (3) Dept. of Microbiology, Ludwig--Maximilians University, Munich, Germany (4) Section of Clinical Immunology, Dept. of Internal Medicine, University Hospital, Zurich, Switzerland Article History: Registration Date: 01/01/2005 Received Date: 23/06/2004 Accepted Date: 24/11/2004 Online Date: 06/03/2005 Article note: Presented on the 14th Meeting of the European Neurological Society (Barcelona, Spain, 26--30 June, 2004). Informed consent for lumbar puncture was obtained from all patients. There was no special financial support. The authors have no conflict of interest

Swimming bacteria detect chemical gradients by performing temporal comparisons of recent measurements of chemical concentration. These comparisons are described quantitatively by the chemotactic response function, which we expect to optimize chemotactic behavioral performance. We identify two independent chemotactic performance criteria: In the short run, a favorable response function should move bacteria up chemoattractant gradients; in the long run, bacteria should aggregate at peaks of chemoattractant concentration. Surprisingly, these two criteria conflict, so that when one performance criterion is most favorable, the other is unfavorable. Because both types of behavior are biologically relevant, we include both behaviors in a composite optimization that yields a response function that closely resembles experimental measurements. Our work suggests that the bacterial chemotactic response function can be derived from simple behavioral considerations and sheds light on how the response function contributes to chemotactic performance. chemotaxis | optimization | strategy

Bacterial chemotaxis is said to have a significant impact on the structure and function of bacterial communities. According to the experiments performed, the results demonstrate that the continuous flow apparatus accurately measures diffusive transport.

Myxococcus xanthus genetic mutatants with characterized phenotypes were analysed for the ability to prey on susceptible bacteria. Quantification of predatory ability was scored by a newly developed method under conditions in which prey bacteria provided the only source of nutrients. These results were corroborated by data derived using a previously published protocol that measures predation in the presence of limited external nutrients. First, early developmental regulatory mutants were examined, because their likely functions in assessing the local nutrient status were predicted to be also important for predation. The results showed that predation efficiency is reduced by 64-80% for mutants of three A-signalling components, AsgA, AsgC and AsgE, but not for AsgB. This suggests that an Asg regulon function that is separate from A-signal production is needed for predation. Besides the Asg components, mutations in the early developmental genes sdeK and csgA were also consistently observed to reduce predatory efficacy by 36 and 33%, respectively. In contrast, later developmental components, such as DevRS, 4406 and PhoP4, did not appear to play significant roles in predation. The predatory abilities of mutants defective for motility were also tested. The data showed that adventurous, but not social, motility is required for predation in the assay. Also, mutants for components in the chemotaxis-like Frz system were found to be reduced in predation efficiency by between 62 and 85%. In sum, it was demonstrated here that defects in development and development-related processes affect the ability of M. xanthus to prey on other bacteria.

Motile bacteria employ sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioural responses. The proteins involved in this signalling pathway have been extensively studied on a molecular level in various model organisms, including enterobacteria and Bacillus subtilis, and specific protein--protein interactions have been identified. The chemotaxis operon of spirochaetes encodes a novel chemotaxis protein, CheX, in addition to homologues to the central components of established chemotaxis systems. Interestingly, the closest functionally characterized homologue of CheX is CheC of the complex B. subtilis chemotaxis pathway. In this study, the yeast two-hybrid system was applied to investigate protein-protein interactions within the chemotaxis signalling pathway of Treponema denticola, with special focus on CheX. CheX was found to interact with CheA and with itself. The other chemotaxis proteins exhibited interactions comparable to their homologues in known chemotaxis systems. Based on these findings, a model integrating CheX in the chemotaxis signal transduction pathway of T. denticola is proposed.

The systemic vasculature in and surrounding the lung is proangiogenic, whereas the pulmonary vasculature rarely participates in neovascularization. We studied the effects of the proangiogenic ELR+ CXC chemokine MIP-2 (macrophage inflammatory protein-2) on endothelial cell proliferation and chemotaxis. Mouse aortic, pulmonary arterial, and lung microvascular endothelial cells were isolated and subcultured. Proliferation ([[.sup.3]H]thymidine uptake) and migration (Transwell chemotaxis) were evaluated in each cell type at baseline and upon exposure to MIP-2 (1-100 ng/ml) without and with exposure to hypoxia (24 h)-reoxygenation. Baseline proliferation did not vary among cell types, and all cells showed increased proliferation after MIP-2. Aortic cell chemotaxis increased markedly upon exposure to MIP-2; however, neither pulmonary artery nor lung microvascular endothelial cells responded to this chemokine. Assessment of CXC[R.sub.2], the G protein-coupled receptor through which MIP-2 signals, displayed no baseline difference in mRNA, protein, or cell surface expression among cell types. Exposure to hypoxia increased expression of CXC[R.sub.2] of aortic endothelial cells only. Additionally, aortic cells, compared with pulmonary cells, showed significantly greater protein and activity of cathepsin S, a proteolytic enzyme important for cell motility. Thus the combined effects of increased cathepsin S activity, providing increased motility and enhanced CXC[R.sub.2] expression after hypoxia, both contribute to the proangiogenic phenotype of systemic arterial endothelial cells. angiogenesis; cathepsin S; hypoxia-reoxygenation; macrophage inflammatory protein-2

How do microtubules, which maintain and direct polarity of many eukaryotic cells, regulate polarity of blood neutrophils? In sharp contrast to most cells, disrupting a neutrophil's microtubule network with nocodazole causes it to polarize and migrate [Niggli, V. (2003) J. Cell Sci. 116, 813-822]. Nocodazole induces the same responses in differentiated HL-60 cells, a model neutrophil cell line, and reduces their chemotactic prowess by causing them to pursue abnormally circuitous paths in migrating toward a stationary point source of an attractant, f-Met-Leu-Phe (fMLP). The chemotactic defect stems from dramatic nocodazole-induced imbalance between the divergent, opposed fMLP-induced 'backness' and 'frontness' signals responsible for neutrophil polarity. Nocodazole (i) stimulates backness by increasing Rho- and actomyosindependent contractility, as reported by Niggli, and also (ii) impairs fMLP-dependent frontness: pseudopods are flatter, contain less F-actin, and show decreased membrane translocation of PH-Akt-GFP, a fluorescent marker for 3'-phosphoinositide lipids. Inhibiting backness with a pharmacologic inhibitor of a Rho-dependent kinase substantially reverses nocodazole's effects on chemotaxis, straightness of migration paths, morphology, and PH-Akt-GFP translocation. Thus, microtubules normally balance backness vs. frontness signals, preventing backness from reducing the strength of pseudopods and from impairing directional migration. chemotaxis | PIP3 | pseudopod | Rho GTPases

Flagellate bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium typically express 5 to 12 flagellar filaments over their cell surface that rotate in clockwise (CW) and counterclockwise directions. These bacteria modulate their swimming direction towards favorable environments by biasing the direction of flagellar rotation in response to various stimuli. In contrast, Rhodobacter sphaeroides expresses a single sub-polar flagellum that rotates only CW and responds tactically by a series of biased stops and starts. Rotor protein FliG transiently links the MotAB stators to the rotor, to power rotation and also has an essential function in flagellar export. In this study, we sought to determine whether the FliG protein confers directionality on flagellar motors by testing the functional properties of R. sphaeroides FliG and a chimeric FliG protein, EcRsFliG (N-terminal and central domains of E. coli FliG fused to an R. sphaeroides FliG C terminus), in an E. coli FliG null background. The EcRsFliG chimera supported flagellar synthesis and bidirectional rotation; bacteria swam and tumbled in a manner qualitatively similar to that of the wild type and showed chemotaxis to amino acids. Thus, the FliG C terminus alone does not confer the unidirectional stop-start character of the R. sphaeroides flagellar motor, and its conformation continues to support tactic, switch-protein interactions in a bidirectional motor, despite its evolutionary history in a bacterium with a unidirectional motor.

Delayed polymorphonuclear leukocyte (PMN) apoptosis exacerbates acute lung injury. To reach the alveolar spaces, PMNs must migrate across both pulmonary endothelial and epithelial cell layers. We hypothesized that transmigration across the endothelium-epithelium bilayer suppresses PMN apoptosis and sought to elucidate the underlying mechanisms. PMNs freshly isolated from normal volunteers were allowed to migrate across polycarbonate membranes alone or membranes coated with a bilayer of human lung endothelial and epithelial cells. After migration toward different chemoattractants (IL-8, formy1-Met-Leu-Phe, or leukotriene [B.sub.4]), PMN apoptosis and caspase activities were assessed by annexin V, histology, and enzymatic assays, respectively. Messenger RNA and specific protein expression in three receptor ligand-mediated, apoptosis-inducing pathways (Fas, TNF-[alpha], and TNF-related apoptosis-inducing ligand) were further examined by gene array, RT-PCR, flow cytometry, and Western blot analyses. The data demonstrated that transbilayer migration suppressed PMN apoptosis, and this effect was not chemoattractant type specific. Kinetic analyses further showed that the delay of apoptosis was sustained to at least 18 h. Transbilayer migration caused significant decreases in caspase (-3, -8, and -9) activities. The changes in apoptosis-related gene expression support the survival role of transbilayer migration. Furthermore, the reduced apoptosis was correlated with downregulation of Fas ligand and TNF receptor 1 expression. Our data reveal that migration across a lung endothelium-epithelium bilayer suppresses PMN apoptosis. The decreased activity and/or expression of proapoptotic proteins may provide possible targets for the regulation of inappropriate delay in PMN apoptosis during lung inflammation and injury. neutrophils; chemotaxis; interleukin-8

Intestinal epithelial cell migration plays a key role in gastrointestinal mucosal barrier formation, enterocyte development, differentiation, turnover, wound healing, and adenocarcinoma metastasis. Chemokines, through engagement of their corresponding receptors, are potent mediators of directed cell migration and are critical in the establishment and regulation of innate and adaptive immune responses. The aim of this study was to define the role for the chemokine CXCL12 and its sole cognate receptor CXCR4 in regulating intestinal epithelial cell migration and to determine its impact on barrier integrity. CXCL12 stimulated the dose-dependent chemotactic migration of human T84 colonic epithelial cells. Epithelial cell migration was inhibited by CXCR4 neutralizing antibody, pertussis toxin, LY-294002, and PD-98059, thereby implicating G[[alpha].sub.i], phosphatidylinositol 3-kinase (PI3-kinase), and the ERK1/2 MAP kinase pathways in CXCR4-specific signaling. CXCL12 was also shown to increase barrier integrity, as defined by transepithelial resistance and paracellular flux across differentiating T84 monolayers. To determine whether CXCL12 regulated epithelial restitution, we used the normal nontransformed intestinal epithelial cell-6 (IEC-6) wound healing model. By using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, we first showed expression of both CXCR4 and its ligand by IEC-6 cells. We then demonstrated that CXCL12 activated comparable signaling mechanisms to stimulate epithelial migration in the absence of proliferation in wounded IEC-6 monolayers. Taken together, these data indicate that CXCL12 signaling via CXCR4 directs intestinal epithelial cell migration, harrier maturation, and restitution, consistent with an important mechanistic role for these molecules in mucosal barrier integrity and innate host defense. chemokine; restitution; wound healing; chemotaxis; G protein-coupled receptor

Transmembrane signaling in bacterial chemotaxis has become an important model system for experimental and theoretical studies. These studies have provided a wealth of detailed molecular structures, including the structures of CheA, CheW, and the cytoplasmic domain of the serine receptor Tsr. How these three proteins interact to form the receptor/signaling complex remains unknown. By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor/signaling complex. The complex contains CheA, CheW, and the cytoplasmic portion of the aspartate receptor Tar. We observe density consistent with a structure containing 24 aspartate-receptor monomers and additional density sufficient to house the expected four CheA monomers and six CheW monomers. Within this bipolar structure are four groups of three receptor dimers that are not threefold symmetric and are therefore unlike the symmetric trimers observed in the x-ray crystal structure of the cytoplasmic domain of the serine receptor. In the latter, the interdimer contacts occur in the signaling domains near the hairpin loop. In our structure, the signaling domains within trimers appear spaced apart by the presence of CheA and CheW. This structure argues against models where one CheA and one CheW bind to the outer face of each of the dimers in the trimer. This structure of the receptor/signaling complex provides an additional basis for understanding the architecture of the large arrays of chemotaxis receptors, CheA, and CheW found at the cell poles in motile bacteria. chemotaxis | electron microscopy | macromolecular assembly | signal transduction

Symbioses between zooxanthellae (Symbiodinium spp.) and marine invertebrates, including corals, are common in shallow marine environments. The zooxanthellae contribute to host nutrition by translocating photosynthetic products and enabling them to […]

The chemotactic sensory system of Escherichia coli comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins. These components form signaling complexes that mediate sensory excitation and adaptation. Previous determinations of cellular content of individual components provided differing and apparently conflicting values. We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two E. coli strains considered wild type for chemotaxis, grown in rich and minimal media. Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30%. Thus, cellular stoichiometries were almost constant as amounts varied substantially. Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex. The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW. These components may interact in extended arrays and, thus, stoichiometries could be nonintegral. In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively. These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical.