Frozen dynamic dimer model for transmembrane signaling in bacterial chemotaxis receptors.

The crystal structures of the ligand binding domain of a bacterial aspartate receptor suggest a simple mechanism for transmembrane signaling by the dimer of the receptor. On ligand binding, one domain rotates with respect to the other, and this rotational motion is proposed to be transmitted through the membrane to the cytoplasmic domains of the receptor.     

An allosteric model for transmembrane signaling in bacterial chemotaxis

Bacteria are able to sense chemical gradients over a wide range of concentrations. However, calculations based on the known number of receptors do not predict such a range unless receptors interact with one another in a cooperative manner. A number of recent experiments support the notion that this remarkable sensitivity in chemotaxis is mediated by localized interactions or crosstalk between neighboring receptors. A number of simple, elegant models have proposed mechanisms for signal integration within receptor clusters. What is a lacking is a model, based on known molecular mechanisms and our accumulated knowledge of chemotaxis, that integrates data from multiple, heterogeneous sources. To address this question, we propose an allosteric mechanism for transmembrane signaling in bacterial chemotaxis based on the "trimer of dimers" model, where three receptor dimers form a stable complex with CheW and CheA. The mechanism is used to integrate a diverse set of experimental data in a consistent framework. The main predictions are: (1) trimers of receptor dimers form the building blocks for the signaling complexes; (2) receptor methylation increases the stability of the active state and retards the inhibition arising from ligand-bound receptors within the signaling complex; (3) trimer of dimer receptor complexes aggregate into clusters through their mutual interactions with CheA and CheW; (4) cooperativity arises from neighboring interaction within these clusters; and (5) cluster size is determined by the concentration of receptors, CheA, and CheW. The model is able to explain a number of seemingly contradictory experiments in a consistent manner and, in the process, explain how bacteria are able to sense chemical gradients over a wide range of concentrations by demonstrating how signals are integrated within the signaling complex.     

Differences in signalling by directly and indirectly binding ligands in bacterial chemotaxis

In chemotaxis of Escherichia coli and other bacteria, extracellular stimuli are perceived by transmembrane receptors that bind their ligands either directly, or indirectly through periplasmic‐binding proteins (BPs). As BPs are also involved in ligand uptake, they provide a link between chemotaxis and nutrient utilization by cells. However, signalling by indirectly binding ligands remains much less understood than signalling by directly binding ligands. Here, we compared intracellular responses mediated by both types of ligands and developed a new mathematical model for signalling by indirectly binding ligands. We show that indirect binding allows cells to better control sensitivity to specific ligands in response to their nutrient environment and to coordinate chemotaxis with ligand transport, but at the cost of the dynamic range being much narrower than for directly binding ligands. We further demonstrate that signal integration by the chemosensory complexes does not depend on the type of ligand. Overall, our data suggest that the distinction between signalling by directly and indirectly binding ligands is more physiologically important than the traditional distinction between high‐ and low‐abundance receptors.     

Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance‐dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation‐induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation‐dependent and five stimulation‐independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation‐induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.     

Information processing and signal integration in bacterial quorum sensing

Bacteria communicate using secreted chemical signaling molecules called autoinducers in a process known as quorum sensing. The quorum‐sensing network of the marine bacterium Vibrio harveyi uses three autoinducers, each known to encode distinct ecological information. Yet how cells integrate and interpret the information contained within these three autoinducer signals remains a mystery. Here, we develop a new framework for analyzing signal integration on the basis of information theory and use it to analyze quorum sensing in V. harveyi. We quantify how much the cells can learn about individual autoinducers and explain the experimentally observed input–output relation of the V. harveyi quorum‐sensing circuit. Our results suggest that the need to limit interference between input signals places strong constraints on the architecture of bacterial signal‐integration networks, and that bacteria probably have evolved active strategies for minimizing this interference. Here, we analyze two such strategies: manipulation of autoinducer production and feedback on receptor number ratios.     

Signal transduction in neutrophil chemotaxis

This review discusses current knowledge on signal transduction pathways controlling chemotaxis of neutrophils and similar cells. Most neutrophil chemoattractants bind to seven-transmembrane-helix receptors. These receptors activate trimeric G proteins of the Gi class in neutrophils to initiate chemotaxis. Phospholipases Cbeta, phosphoinositide 3-kinase gamma, and PH domain-containing proteins play various roles in signaling further downstream. The actin cytoskeleton is crucial for cell motility, and is controlled by Rho family GTP-binding proteins. PIP 5-kinase, LIM kinase, myosin light chain kinase and phosphatase, or WASP-like proteins may be important links between Rho GTPases and actin during chemotaxis. Newly emerging ideas on the regulation of the "compass" of chemotaxing cells, which may involve Cdc42 and certain PH domain-containing proteins, are also presented.     

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems.     

Extracellular calmodulin: A polypeptide signal in plants?

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca²⁺ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP₃, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.     

Signaling mechanisms for regulation of chemotaxis

Chemotaxis is a fascinating biological process, through which a cell migrates along a shallow chemoattractant gradient that is less than 5% difference between the anterior and posterior of the cell. Chemotaxis is composed of two independent, but interrelated processes-motihty and directionality, both of which are regulated by extracellular stimuli, chemoattractants. In this mini-review, recent progresses in the understanding of the regulation of leukocyte chemotaxis by chemoattractant signaling are reviewed.     

A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.     

In silico tools for signal transduction research

Signal transduction is a fundamental process that takes place in all living organisms and understanding how this event occurs at the cellular level is of vital importance to virtually all fields of biomedicine. There are several major steps involved in deciphering the signalling pathways: (a) Which molecules are involved in signalling? (b) Who talks to whom?, ie making sense of the molecular interactions in a context-dependent way. (c) Where are the signalling events taking place?, eg when a resting cell becomes activated. The challenge lies in reconstructing signalling modules and networks evoked in a particular response to a single input as well as correlating the signalling response to different cellular inputs. There is also the need for interpretation of cross-talk between signalling modules in response to single and multiple inputs. To follow up these questions there are many good databases that provide an information system on regulatory networks. This review aims to find some of the bioinformatics tools and websites available to conduct signal transduction research and to discuss the representation of databases available for the processes of signalling. The databases considered here can provide a well-structured overview on the subject and a basis for advanced bioinformatics analysis to interpret the function of genomic sequences or to analyse signalling networks within a cell. However, the knowledge of most signalling pathways is incomplete and for this reason the existing databases will provide insight, but very rarely a more complete picture.     

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     

细菌群体感应的蛋白质组学研究进展

细菌群体感应是指细菌能合成、释放和感应一些类激素小分子信号,从而调控群体行为并对其作出应答反应。介导细菌群体感应的信号分子有多种,它们参与调节细菌许多重要生物学功能。目前对此研究的主要手段是基因组学和转录组学。然而近年来,基因组测序技术的不断发展为另一种新兴方法——以比较和功能性为基础的蛋白质组学法奠定了基础。所不同的是,传统方法只能局限性研究某些基因或蛋白,而蛋白质组学法能检测出生物体基因表达的全部蛋白,它也因此逐渐受到人们的广泛关注。主要从研究较多的三类信号分子方面描述如何利用蛋白盾组举法解析细菌交流的“语言”。     

Genetic engineering of plant signal transduction mechanisms

Signal transduction is used by plants to coordinate their development and to sense and respond to fluctuations in their surroundings. Of prime importance is the ability to defend against pathogens and other environmental hazards such as cold temperatures, drought or wounding. Many transduction pathways are now characterized and the underlying genes are known. This suggests an obvious question—can we engineer signal transduction mechanisms for plant improvement? We address this question by presenting a rationale for an engineering approach and by discussing results from recent attempts to apply this approach. Calmodulin-like domain protein kinase (CDPK) and mitogen-activated protein kinase (MAPK) pathways are used as primary examples. New technology that will aid these efforts is also covered.     

Quantitative analysis of gradient sensing: towards building predictive models of chemotaxis in cancer

Chemotaxis of tumor cells in response to a gradient of extracellular ligand is an important step in cancer metastasis. The heterogeneity of chemotactic responses in cancer has not been widely addressed by experimental or mathematical modeling techniques. However, recent advancements in chemoattractant presentation, fluorescent-based signaling probes, and phenotypic analysis paradigms provide rich sources for building data-driven relational models that describe tumor cell chemotaxis in response to a wide variety of stimuli. Here we present gradient sensing, and the resulting chemotactic behavior, in a 'cue-signal-response' framework and suggest methods for utilizing recently reported experimental methods in data-driven modeling ventures.     

植物孢子体自交不亲和信号转导功能分子研究进展

孢子体自交不亲和(SSI)是许多植物采取的一种抵制近亲繁殖的重要措施,受S位点复等位基因控制。近年来,参与其信号转导的许多功能分子及它们的编码基因被分离并得到了充分研究：当自花授粉时,SPlI／SCR与SRK特异识别,造成后的Ser／Thr激酶的磷酸化,引发了一系列由SLG、ARC1及水孔蛋白等因子参与的SSI信号转导途径,最终产生自交不亲和的结果。     

同型半胱氨酸对心肌细胞的损伤作用及其信号转导机制探讨

目的：观察同型半胱氨酸（homocysteine,HCY)对心肌细胞的损伤作用,探讨该作用发生的信号转导机制及其关键调控环节。方法：分离培养Wistar乳鼠心肌细胞,经HCY作用后,以台盼蓝排斥实验测定细胞存活率,TUNEL法和流式细胞仪测定细胞凋亡率,免疫印迹法测定心肌细胞ERK2蛋白磷酸化水平,阻滞电泳法测定细胞NF－κB活化水平。结果：作用后,心肌细胞存活率显著降低,存活率降低程度与HCY的作用浓度、作用时间具有明确的剂量－效应关系及时间－效应关系;在10^-3mol/L HCY作用下,心肌细胞凋亡率升高,于4h达峰值,为7．56％;10^-3,10^-4、10^-5mol/L HCY均可抑制心肌细胞ERK2磷酸化,其中10^-3mol/L HCY作用于心肌细胞后,ERYK2蛋白磷酸化水平呈现迅速而明显的降低,4h降至对照组的3．04％（P＜0．01）;不同浓度HCY均明显阻抑NF－κB的活化。结论：HCY具有明显心肌细胞损伤作用,对心肌细胞凋亡的诱导是HCY心肌细胞损伤作用的形式之一;HCY可影响心肌细胞信号转导通路ERK,通过对转录因子NF－κB的活化抑制,导致心肌细胞损伤。     

STAT1 is involved in signal transduction in the EPO induced HEL cells

Erythropoietin(EPO) is the major regulator of mamalian erythropoisis,which stimulates the growth and differentiation of hematopoietic cells through interaction with its receptor(EPO-R),Here we use HEL cells (a human erythro-leukemia cell line) as a model to elucidate the pathway of signal transduction in the EPO-induced HEL cells.Our data show that the EPOR (EPO receptor) on the surface of HEL cells interacts with the Janus tyrosine protein kinase(Jak2) to transduce intracellular signals through phosphorylation of cytoplasmic proteins in EPO-treated HEL cells.Both STAT1 and STAT5 in this cell line are tyrosine-phosphorylated and translocated to nucleus following the dinding of EPO to HEL cells.Furthermore,the dinding of both STAT1 and STAT5 proteins to specific DNA elements(SIE and PIE elements) is revealed in an EPO-dependent manner,Our data demonstrate that the pathway of signal transduction following the binding of EPO to HEL cells is similar to immature eryhroid cell from the spleen of mice infected with anemia strain of Friend virus.     

Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold

Initiation of cell migration requires morphological polarization with formation of a dominant leading pseudopodium and rear compartment. A molecular understanding of this process has been limited, due to the inability to biochemically separate the leading pseudopodium from the rear of the cell. Here we examine the spatio-temporal localization and activation of cytoskeletal-associated signals in purified pseudopodia directed to undergo growth or retraction. Pseudopodia growth requires assembly of a p130Crk-associated substrate (CAS)/c-CrkII (Crk) scaffold, which facilitates translocation and activation of Rac1. Interestingly, Rac1 activation then serves as a positive-feedback loop to maintain CAS/Crk coupling and pseudopodia extension. Conversely, disassembly of this molecular scaffold is critical for export and down regulation of Rac1 activity and induction of pseudopodia retraction. Surprisingly, the uncoupling of Crk from CAS during pseudopodium retraction is independent of changes in focal adhesion kinase activity and CAS tyrosine phosphorylation. These findings establish CAS/Crk as an essential scaffold for Rac1-mediated pseudopodia growth and retraction, and illustrate spatio-temporal segregation of cytoskeletal signals during cell polarization.