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

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

A framework for mapping,visualisation and automatic model creation of signal‐transduction networks

Intracellular signalling systems are highly complex. This complexity makes handling, analysis and visualisation of available knowledge a major challenge in current signalling research. Here, we present a novel framework for mapping signal‐transduction networks that avoids the combinatorial explosion by breaking down the network in reaction and contingency information. It provides two new visualisation methods and automatic export to mathematical models. We use this framework to compile the presently most comprehensive map of the yeast MAP kinase network. Our method improves previous strategies by combining (I) more concise mapping adapted to empirical data, (II) individual referencing for each piece of information, (III) visualisation without simplifications or added uncertainty, (IV) automatic visualisation in multiple formats, (V) automatic export to mathematical models and (VI) compatibility with established formats. The framework is supported by an open source software tool that facilitates integration of the three levels of network analysis: definition, visualisation and mathematical modelling. The framework is species independent and we expect that it will have wider impact in signalling research on any system.     

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway

Large‐scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi‐dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein–protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA‐mediated gene knockdown on Wnt signaling. Meta‐analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates β‐catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.     

Stimulation of the yeast high osmolarity glycerol (HOG) pathway: evidence for a signal generated by a change in turgor rather than by water stress

The Saccharomyces cerevisiae HOG pathway controls responses to osmotic shock such as production of the osmolyte glycerol. Here we show that the HOG pathway can be stimulated by addition of glycerol. This stimulation was strongly diminished in cells expressing an unregulated Fps1p glycerol channel, presumably because glycerol rapidly equilibrated across the plasma membrane. Ethanol, which passes the plasma membrane readily and causes water stress by disturbing the hydration of biomolecules, did not activate the HOG pathway. These observations suggest that stimulation of the HOG pathway is mediated by a turgor change and not by water stress per se.     

Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB‐VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB‐VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved‐signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.     

High osmolarity glycerol (HOG) pathway-induced phosphorylation and activation of 6-phosphofructo-2-kinase are essential for glycerol accumulation and yeast cell proliferation under hyperosmotic stress

In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation. The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades. The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure. We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells. A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme. An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value. This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway. In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser(644) and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt. The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway. The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation. Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.     

Co‐ordination of osmotic stress responses through osmosensing and signal transduction events in fishes

This review centres upon the molecular regulation of osmotic stress responses in fishes, focusing on how osmosensing and signal transduction events co‐ordinate changes in the activity and abundance of effector proteins during osmotic stress and how these events integrate into osmotic stress responses of varying magnitude. The concluding sections discuss the relevance of osmosensory signal transduction to the evolution of euryhalinity and present experimental approaches that may best stimulate future research. Iterating the importance of osmosensing and signal transduction during fish osmoregulation may be pertinent amidst the increased use of genomic technologies that typically focus solely on changes in the abundances of gene products, and may limit insight into critical upstream events that occur mainly through post‐translational mechanisms.     

Role of the JAK/STAT signal transduction pathway in the regulation of gene expression in CNS

Over the last 20 years the JAK/STAT signal transduction pathway has been extensively studied. An enormous amount of data on different cell signal transduction pathways is now available. The JAK/STAT signal transduction pathway is one of the intracellular signaling pathways activated by cytokines and growth factors that was first studied in the hematopoietic system, but recent data demonstrate that this signal transduction is also greatly utilized by other systems. The JAK/STAT pathway is a signaling cascade that links the activation of specific cell membrane receptors to nuclear gene expression. This review is focused on the role of JAK/STAT signal transduction pathway activation in the central nervous system (CNS).     

果蝇背闭合行为中DJNK信号转导途径研究进展

Jun氨基末端激酶 (JunN terminalkinase ,JNK)是一种重要的细胞信号传递者。它参与了细胞生长、分化、程序性死亡等生理过程 ,而且在调节上皮细胞运动和形态发生等方面也起着重要作用。大量研究证实 ,在果蝇Drosophila的背闭合行为 (dorsalclosure,DC)中 ,DJNK(DrosophilaJNK)的调节是关键。文章就果蝇DC的发生过程以及DJNK信号途径的研究进展作一简要的综述。     

Interaction between the Candida albicans high-osmolarity glycerol (HOG) pathway and the response to human beta-defensins 2 and 3

Human β-defensins 2 and 3 are small cationic peptides with antimicrobial activity against the fungal pathogen Candida albicans. We found that hog1 and pbs2 mutants were hypersensitive to treatment with these peptides, pointing to a role of the high-osmolarity glycerol (HOG) pathway in the response to defensin-induced cell injury.     

Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions

We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.     

Antigen‐responsive regulation of Cell motility and migration via the signalobodies based on c‐Fms and c‐Mpl

Since cell migration plays critical roles in development and homeostasis of the body, artificial control of cell migration would be promising for the treatment of various diseases related to migration. To this end, we previously developed single‐chain Fv (scFv)/receptor chimeras, named signalobodies, which can control cell fates via a specific antigen that is different from natural cytokines. Although a conventional chemotaxis chamber assay revealed that several signalobodies based on receptor tyrosine kinases transduced antigen‐dependent migration signals, we have never performed direct observation of the cells to obtain more information on overall properties of cell motility and migration. In this study, we utilized murine pro‐B Ba/F3 cells expressing either a scFv‐Fms or scFv‐Mpl signalobody, and compared their migratory characteristics. We employed a lipid–polyethylene glycol conjugate to softly immobilize the suspension cells on a slide, which facilitated direct observation of chemokinetic activity of the cells. Consequently, both cells markedly exhibited chemokinesis in response to a specific antigen. In addition, the cells were subjected to a stable antigen‐concentration gradient to observe horizontal directional cell migration in real time. The results showed that the cells expressing scFv‐Fms underwent directional migration toward a positive antigen‐concentration gradient. Taken together, we successfully demonstrated antigen‐responsive regulation of cell motility and migration via the signalobodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:411–417, 2014     

Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions

Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin-like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA-dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet-mutants have been isolated which showed a constitutive expression of the thionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1-GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12-oxophytodienoic acid (OPDA) up to 40-fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)- and the JA-dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet-mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.     

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.