Signaling Pathways in Bacterial Chemotaxis

The sensory transduction pathways between the transducing proteins and the switch on the flagellar motors have been investigated in Escherichia coli and Salmonella typhimurium. ATP, not GTP, is required for normal chemotaxis. A site of ATP action appears to be the conversion of an inactive form of the CheY protein to an active form, designated CheY*, that binds to the motor switch and initiates clockwise rotation. The methylation-dependent and methylation-independent pathways for chemotaxis have a common requirement for the CheA, CheW, and CheY proteins in addition to the switch and flagellar motor. It is concluded that the receptor/transducing proteins and the adaptation mechanism differ in the two types of pathway, but that other components of the transduction pathway are common to the methylation-dependent and methylation-independent pathways.     

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.     

Dopamine Modulates Persistent Synaptic Activity and Enhances the Signal-to-Noise Ratio in the Prefrontal Cortex

Background

The importance of dopamine (DA) for prefrontal cortical (PFC) cognitive functions is widely recognized, but its mechanisms of action remain controversial. DA is thought to increase signal gain in active networks according to an inverted U dose-response curve, and these effects may depend on both tonic and phasic release of DA from midbrain ventral tegmental area (VTA) neurons.

Methodology/Principal Findings

We used patch-clamp recordings in organotypic co-cultures of the PFC, hippocampus and VTA to study DA modulation of spontaneous network activity in the form of Up-states and signals in the form of synchronous EPSP trains. These cultures possessed a tonic DA level and stimulation of the VTA evoked DA transients within the PFC. The addition of high (≥1 µM) concentrations of exogenous DA to the cultures reduced Up-states and diminished excitatory synaptic inputs (EPSPs) evoked during the Down-state. Increasing endogenous DA via bath application of cocaine also reduced Up-states. Lower concentrations of exogenous DA (0.1 µM) had no effect on the up-state itself, but they selectively increased the efficiency of a train of EPSPs to evoke spikes during the Up-state. When the background DA was eliminated by depleting DA with reserpine and alpha-methyl-p-tyrosine, or by preparing corticolimbic co-cultures without the VTA slice, Up-states could be enhanced by low concentrations (0.1–1 µM) of DA that had no effect in the VTA containing cultures. Finally, in spite of the concentration-dependent effects on Up-states, exogenous DA at all but the lowest concentrations increased intracellular current-pulse evoked firing in all cultures underlining the complexity of DA''s effects in an active network.

Conclusions/Significance

Taken together, these data show concentration-dependent effects of DA on global PFC network activity and they demonstrate a mechanism through which optimal levels of DA can modulate signal gain to support cognitive functioning.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.     

Characterization of the Adaptation Module of the Signaling Network in Bacterial Chemotaxis by Measurement of Step Responses

The bacterial chemotaxis network features robust adaptation implemented by negative integral feedback. Here, we show that the adaptation module can be characterized by measurement of the response to simple step-addition and removal of a chemoattractant. The method does not rely on a particular form of the receptor module, and thus can be used to characterize other integral feedback networks.     

Heparanase Enhances the Insulin Receptor Signaling Pathway to Activate Extracellular Signal-regulated Kinase in Multiple Myeloma

ERK signaling regulates proliferation, survival, drug resistance, and angiogenesis in cancer. Although the mechanisms regulating ERK activation are not fully understood, we previously demonstrated that ERK phosphorylation is elevated by heparanase, an enzyme associated with aggressive behavior of many cancers. In the present study, myeloma cell lines expressing either high or low levels of heparanase were utilized to determine how heparanase stimulates ERK signaling. We discovered that the insulin receptor was abundant on cells expressing either high or low levels of heparanase, but the receptor was highly phosphorylated in heparanase-high cells compared with heparanase-low cells. In addition, protein kinase C activity was elevated in heparanase-high cells, and this enhanced expression of insulin receptor substrate-1 (IRS-1), the principle intracellular substrate for phosphorylation by the insulin receptor. Blocking insulin receptor function with antibody or a small molecule inhibitor or knockdown of IRS-1 expression using shRNA diminished heparanase-mediated ERK activation in the tumor cells. In addition, up-regulation of the insulin signaling pathway by heparanase and the resulting ERK activation were dependent on heparanase retaining its enzyme activity. These results reveal a novel mechanism whereby heparanase enhances activation of the insulin receptor signaling pathway leading to ERK activation and modulation of myeloma behavior.     

Universal Response-Adaptation Relation in Bacterial Chemotaxis

The bacterial strategy of chemotaxis relies on temporal comparisons of chemical concentrations, where the probability of maintaining the current direction of swimming is modulated by changes in stimulation experienced during the recent past. A short-term memory required for such comparisons is provided by the adaptation system, which operates through the activity-dependent methylation of chemotaxis receptors. Previous theoretical studies have suggested that efficient navigation in gradients requires a well-defined adaptation rate, because the memory time scale needs to match the duration of straight runs made by bacteria. Here we demonstrate that the chemotaxis pathway of Escherichia coli does indeed exhibit a universal relation between the response magnitude and adaptation time which does not depend on the type of chemical ligand. Our results suggest that this alignment of adaptation rates for different ligands is achieved through cooperative interactions among chemoreceptors rather than through fine-tuning of methylation rates for individual receptors. This observation illustrates a yet-unrecognized function of receptor clustering in bacterial chemotaxis.     

Protein Connectivity in Chemotaxis Receptor Complexes

The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.     

Role of Methionine in Bacterial Chemotaxis

The effect of methionine starvation on the motility and behavior of normal and nonchemotactic mutants of Salmonella typhimurium was investigated. Methionine starvation eliminates tumbling in the wild type, but fails to do so in an uncoordinated (frequent tumbling) mutant. In the mutant, methionine starvation significantly extends the period of smooth motility that follows a sharp temporal increase of attractant. These results suggest that methionine metabolism is not tightly coupled to the generation of tumbles, but rather is necessary for the return of some tumble-regulating parameter to a steady-state level.     

CheA-Receptor Interaction Sites in Bacterial Chemotaxis

In bacterial chemotaxis, transmembrane chemoreceptors, the CheA histidine kinase, and the CheW coupling protein assemble into signaling complexes that allow bacteria to modulate their swimming behavior in response to environmental stimuli. Among the protein-protein interactions in the ternary complex, CheA-CheW and CheW-receptor interactions were studied previously, whereas CheA-receptor interaction has been less investigated. Here, we characterize the CheA-receptor interaction in Thermotoga maritima by NMR spectroscopy and validate the identified receptor binding site of CheA in Escherichia coli chemotaxis. We find that CheA interacts with a chemoreceptor in a manner similar to that of CheW, and the receptor binding site of CheA's regulatory domain is homologous to that of CheW. Collectively, the receptor binding sites in the CheA-CheW complex suggest that conformational changes in CheA are required for assembly of the CheA-CheW-receptor ternary complex and CheA activation.     

Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca²⁺ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.     

Microenvironmental Stiffness Enhances Glioma Cell Proliferation by Stimulating Epidermal Growth Factor Receptor Signaling

The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling.     

Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.     

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly ¹. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells ^{2, 3}. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo⁴. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells ^5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits ^{7, 9, 10}. Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP₂ into PIP₃ on the cell membrane ^11-13. PIP₃ serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane ^{14, 15}. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP₃ to PIP₂^{16, 17}. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils ¹⁸. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP₃ responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.Download video file.^{(59M, mov)}     

Quantitative Analysis of Bacterial Migration in Chemotaxis

THERE are a number of parallels between the chemotactic response of bacteria and the process of chemoreception in higher organisms^{1, 2}. Because of this analogy, the chemotactic response presents an unusual opportunity in a simple system for delineating some of the features of a receptor system. Adler and co-workers^3–5, for example, have made particularly important advances in studies on Escherichia coli which indicate that (a) the galactose binding protein^{6, 7} is the receptor which triggers the chemotactic response⁵ and (b) the response occurs with non-metabolizableanalogues of galactose³.     

Limits of Feedback Control in Bacterial Chemotaxis

Inputs to signaling pathways can have complex statistics that depend on the environment and on the behavioral response to previous stimuli. Such behavioral feedback is particularly important in navigation. Successful navigation relies on proper coupling between sensors, which gather information during motion, and actuators, which control behavior. Because reorientation conditions future inputs, behavioral feedback can place sensors and actuators in an operational regime different from the resting state. How then can organisms maintain proper information transfer through the pathway while navigating diverse environments? In bacterial chemotaxis, robust performance is often attributed to the zero integral feedback control of the sensor, which guarantees that activity returns to resting state when the input remains constant. While this property provides sensitivity over a wide range of signal intensities, it remains unclear how other parameters such as adaptation rate and adapted activity affect chemotactic performance, especially when considering that the swimming behavior of the cell determines the input signal. We examine this issue using analytical models and simulations that incorporate recent experimental evidences about behavioral feedback and flagellar motor adaptation. By focusing on how sensory information carried by the response regulator is best utilized by the motor, we identify an operational regime that maximizes drift velocity along chemical concentration gradients for a wide range of environments and sensor adaptation rates. This optimal regime is outside the dynamic range of the motor response, but maximizes the contrast between run duration up and down gradients. In steep gradients, the feedback from chemotactic drift can push the system through a bifurcation. This creates a non-chemotactic state that traps cells unless the motor is allowed to adapt. Although motor adaptation helps, we find that as the strength of the feedback increases individual phenotypes cannot maintain the optimal operational regime in all environments, suggesting that diversity could be beneficial.     

Ecology and Physics of Bacterial Chemotaxis in the Ocean

Summary: Intuitively, it may seem that from the perspective of an individual bacterium the ocean is a vast, dilute, and largely homogeneous environment. Microbial oceanographers have typically considered the ocean from this point of view. In reality, marine bacteria inhabit a chemical seascape that is highly heterogeneous down to the microscale, owing to ubiquitous nutrient patches, plumes, and gradients. Exudation and excretion of dissolved matter by larger organisms, lysis events, particles, animal surfaces, and fluxes from the sediment-water interface all contribute to create strong and pervasive heterogeneity, where chemotaxis may provide a significant fitness advantage to bacteria. The dynamic nature of the ocean imposes strong selective pressures on bacterial foraging strategies, and many marine bacteria indeed display adaptations that characterize their chemotactic motility as “high performance” compared to that of enteric model organisms. Fast swimming speeds, strongly directional responses, and effective turning and steering strategies ensure that marine bacteria can successfully use chemotaxis to very rapidly respond to chemical gradients in the ocean. These fast responses are advantageous in a broad range of ecological processes, including attaching to particles, exploiting particle plumes, retaining position close to phytoplankton cells, colonizing host animals, and hovering at a preferred height above the sediment-water interface. At larger scales, these responses can impact ocean biogeochemistry by increasing the rates of chemical transformation, influencing the flux of sinking material, and potentially altering the balance of biomass incorporation versus respiration. This review highlights the physical and ecological processes underpinning bacterial motility and chemotaxis in the ocean, describes the current state of knowledge of chemotaxis in marine bacteria, and summarizes our understanding of how these microscale dynamics scale up to affect ecosystem-scale processes in the sea.     

The HPV16 E6 Oncoprotein Causes Prolonged Receptor Protein Tyrosine Kinase Signaling and Enhances Internalization of Phosphorylated Receptor Species

The high-risk human papillomavirus (HPV) E6 proteins are consistently expressed in HPV-associated lesions and cancers. HPV16 E6 sustains the activity of the mTORC1 and mTORC2 signaling cascades under conditions of growth factor deprivation. Here we report that HPV16 E6 activated mTORC1 by enhanced signaling through receptor protein tyrosine kinases, including epidermal growth factor receptor and insulin receptor and insulin-like growth factor receptors. This is evidenced by sustained signaling through these receptors for several hours after growth factor withdrawal. HPV16 E6 increased the internalization of activated receptor species, and the signaling adaptor protein GRB2 was shown to be critical for HPV16 E6 mediated enhanced EGFR internalization and mTORC1 activation. As a consequence of receptor protein kinase mediated mTORC1 activation, HPV16 E6 expression increased cellular migration of primary human epithelial cells. This study identifies a previously unappreciated mechanism by which HPV E6 proteins perturb host-signaling pathways presumably to sustain protein synthesis during the viral life cycle that may also contribute to cellular transforming activities of high-risk HPV E6 proteins.