Models of Eukaryotic Gradient Sensing: Application to Chemotaxis of Amoebae and Neutrophils

Eukaryotic cells can detect shallow gradients of chemoattractants with exquisite precision and respond quickly to changes in the gradient steepness and direction. Here, we describe a set of models explaining both adaptation to uniform increases in chemoattractant and persistent signaling in response to gradients. We demonstrate that one of these models can be mapped directly onto the biochemical signal-transduction pathways underlying gradient sensing in amoebae and neutrophils. According to this scheme, a locally acting activator (PI3-kinase) and a globally acting inactivator (PTEN or a similar phosphatase) are coordinately controlled by the G-protein activation. This signaling system adapts perfectly to spatially homogeneous changes in the chemoattractant. In chemoattractant gradients, an imbalance between the action of the activator and the inactivator results in a spatially oriented persistent signaling, amplified by a substrate supply-based positive feedback acting through small G-proteins. The amplification is activated only in a continuous presence of the external signal gradient, thus providing the mechanism for sensitivity to gradient alterations. Finally, based on this mapping, we make predictions concerning the dynamics of signaling. We propose that the underlying principles of perfect adaptation and substrate supply-based positive feedback will be found in the sensory systems of other chemotactic cell types.     

Dynamic Regulation of Novel and Conserved miRNAs Across Various Tissues of Diverse Cucurbit Species

MicroRNA genes (miRNAs) encoding small non-coding RNAs are abundant in plant genomes and play a key role in regulating several biological mechanisms. Five conserved miRNAs, miR156, miR168-1, miR168-2, miR164, and miR166 were selected for analysis from the 21 known plant miRNA families that were recovered from deep sequencing data of small RNA libraries of pumpkin and squash. A total of six novel miRNAs that were not reported before were found to have precursors with reliable fold-back structures and hence considered novel and were designated as cuc_nov_miRNAs. A set of five conserved, six novel miRNAs, and five uncharacterized small RNAs from the deep sequencing data were profiled for their dynamic regulation using qPCR. The miRNAs were evaluated for differential regulation across the tissues among four diverse cucurbit species, including pumpkin and squash (Cucurbita moschata Duch. Ex Poir. and Cucurbita pepo L.), bitter melon (Momordica charantia L.), and Luffa (Loofah) (Luffa acutangula Roxb.). Expression analysis revealed differential regulation of various miRNAs in leaf, stem, and fruit tissues. Importantly, differences in the expression levels were also found in the leaves and fruits of closely related C. moschata and C. pepo. Comparative miRNA profiling and expression analysis in four cucurbits led to identification of conserved miRNAs in cucurbits. Predicted targets for two of the conserved miRNAs suggested miRNAs are involved in regulating similar biological mechanisms in various species of cucurbits.     

Ageing in Plants: Conserved Strategies and Novel Pathways

Abstract: Ageing increases chaos and entropy and ultimately leads to the death of living organisms. Nevertheless, single gene mutations substantially alter lifespan, revealing that ageing is subject to genetic control. In higher plants, ageing is most obviously manifested by the senescence of leaves, and recent molecular genetic studies, in particular the isolation of Arabidopsis mutants with altered leaf senescence, have greatly advanced our understanding of ageing regulation in plants. This paper provides an overview of the identified genes and their respective molecular pathways. Hormones, metabolic flux, reactive oxygen species and protein degradation are prominent strategies employed by plants to control leaf senescence. Plants predominantly use similar ageing-regulating strategies as yeast and animals but have evolved different molecular pathways. The senescence window concept is proposed to describe the age-dependent actions of the regulatory genes. It is concluded that the similarities and differences in ageing between plants and other organisms are deeply rooted in the evolution of ageing and we hope to stimulate discussion and research in the fascinating field of leaf senescence.     

Dependence of Bacterial Chemotaxis on Gradient Shape and Adaptation Rate

Simulation of cellular behavior on multiple scales requires models that are sufficiently detailed to capture central intracellular processes but at the same time enable the simulation of entire cell populations in a computationally cheap way. In this paper we present RapidCell, a hybrid model of chemotactic Escherichia coli that combines the Monod-Wyman-Changeux signal processing by mixed chemoreceptor clusters, the adaptation dynamics described by ordinary differential equations, and a detailed model of cell tumbling. Our model dramatically reduces computational costs and allows the highly efficient simulation of E. coli chemotaxis. We use the model to investigate chemotaxis in different gradients, and suggest a new, constant-activity type of gradient to systematically study chemotactic behavior of virtual bacteria. Using the unique properties of this gradient, we show that optimal chemotaxis is observed in a narrow range of CheA kinase activity, where concentration of the response regulator CheY-P falls into the operating range of flagellar motors. Our simulations also confirm that the CheB phosphorylation feedback improves chemotactic efficiency by shifting the average CheY-P concentration to fit the motor operating range. Our results suggest that in liquid media the variability in adaptation times among cells may be evolutionary favorable to ensure coexistence of subpopulations that will be optimally tactic in different gradients. However, in a porous medium (agar) such variability appears to be less important, because agar structure poses mainly negative selection against subpopulations with low levels of adaptation enzymes. RapidCell is available from the authors upon request.     

The Signaling Mechanisms Underlying Cell Polarity and Chemotaxis

Chemotaxis—the directed movement of cells in a gradient of chemoattractant—is essential for neutrophils to crawl to sites of inflammation and infection and for Dictyostelium discoideum (D. discoideum) to aggregate during morphogenesis. Chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. Extensive studies have been devoted to achieving a better understanding of the mechanism(s) used by a neutrophil to choose its direction of polarity and to crawl effectively in response to chemoattractant gradients. Recent technological advances are beginning to reveal many fascinating details of the intracellular signaling components that spatially direct the cytoskeleton of neutrophils and D. discoideum and the complementary mechanisms that make the cell''s front distinct from its back.Chemotaxis—the directed movement of cells in a gradient of chemoattractant—allows leukocytes to seek out sites of inflammation and infection, amoebas of Dictyostelium discoideum (D. discoideum) to aggregate, neurons to send projections to specific regions of the brain to find their synaptic partners, yeast cells to mate, and fibroblasts to move into the wound space (Fig. 1). In each case, chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. During chemotaxis, filamentous actin (F-actin) is polymerized asymmetrically at the upgradient edge of the cell (leading edge), providing the necessary force to thrust projections of the plasma membrane in the proper direction (see Mullins 2009). Neutrophilic leukocytes (neutrophils), for instance, can polarize and move up very shallow gradients, with a chemoattractant concentration ∼2% higher at the front than the back (Fig. 2) (Devreotes and Zigmond 1988). To restrict actin polymerization to the leading edge in such a shallow gradient, neutrophils must create a much steeper internal gradient of regulatory signals. In addition, distinctive actin–myosin contractile complexes are also formed at the sides and back of the cells (Fig. 2). The ability to create such distinctive segregation of actin assemblies enables neutrophils to move nearly 50 times more quickly than fibroblasts. The polarization response is self-organizing, which occurs even when the attractant concentration is uniform and apparently stimulating all portions of the plasma membrane at the same intensity; in the absence of a gradient, the direction of polarity is random, but all cells can be induced to polarize (Fig. 2). Thus, neutrophil polarization to chemoattractant stimulation represents a striking example of symmetry breaking from an unpolarized state to a polarized one.Open in a separate windowFigure 1.Examples of chemotaxis. (A) A human neutrophil chasing a Staphylococcus aureus microorganism on a blood film among red blood cells, notable for their dark color and principally spherical shape (imaged by David Rogers, courtesy of Thomas P. Stossel). Bar, 10 µm. Chemotaxis is also necessary for (B) D. discoideum to form multicellular aggregates during development (courtesy of M.J. Grimson and R.L. Blanton, Texas Tech University), and (C) for axons to find their way in the developing nervous system. Photo provided by Kathryn Tosney, University of Miami.Open in a separate windowFigure 2.(A–D) Polarization of a neutrophil in response to gradient of chemoattractant. Nomarski images of unpolarized neutrophil responding to a micropipette containing the chemoattractant fMLP (white circle) at (A) 5 s, (B) 30 s, (C) 81 s, and (D) 129 s of stimulation. Bar = 5 µm. (Figure is taken from Weiner et al. 1999, with permission.) Human neutrophils stimulated with fMLP show highly polarized morphology and asymmetric cytoskeletal assemblies. (E–G) Human neutrophils were stimulated by a uniform concentration of fMLP (100 nM) and fixed 2 min after stimulations. Fixed cells were stained for F-actin with rhodamine-phalloidin (E, red) and an antibody raised against activated myosin II (phosphorylated specifically at Ser19, p[19]-MLC) (F, green). These fluorescent images are merged with Nomarski image in (G). Bars, 10 µm.To enter an infected tissue, neutrophils require chemoattractants produced by host cells and microorganisms to migrate to the sites and infection and inflammation. Neutrophil chemotaxis also contributes to many inflammatory and autoimmune diseases, including rheumatoid arthritis, ischemia-reperfusion syndrome, acute respiratory distress, and systemic inflammatory response syndromes. Although the critical physiological functions of neutrophils have made their chemoattractants and chemoattractant receptors targets of intense investigation, understanding of the neutrophil polarity and directional migration has until recently lagged behind that of other cells. Over the past decade, experimentation with knockout mice and human neutrophil cell lines has begun to shed light on the complex intracellular signals responsible for neutrophil polarity. In this article, I summarize recent advances in the study of chemotactic signals in neutrophils, with some of the discussion also devoted to a related model—chemotaxis of D. discoideum. These soil amoebas grow as single cells, but on starvation chemotax into multicellular aggregates in response to secreted chemoattractants such as adenosine 3′,5′-monophosphate (cAMP).     

Diverse Mechanisms of RNA Recombination

Recombination is widespread among RNA viruses, but many molecular mechanisms of this phenomenon are still poorly understood. It was believed until recently that the only possible mechanism of RNA recombination is replicative template switching, with synthesis of a complementary strand starting on one viral RNA molecule and being completed on another. The newly synthesized RNA is a primary recombinant molecule in this case. Recent studies have revealed other mechanisms of replicative RNA recombination. In addition, recombination between the genomes of RNA viruses can be nonreplicative, resulting from a joining of preexisting parental molecules. Recombination is a potent tool providing for both the variation and conservation of the genome in RNA viruses. Replicative and nonreplicative mechanisms may contribute differently to each of these evolutionary processes. In the form of trans splicing, nonreplicative recombination of cell RNAs plays an important role in at least some organisms. It is conceivable that RNA recombination continues to contribute to the evolution of DNA genomes.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 618–632.Original Russian Text Copyright © 2005 by Gmyl, Agol.     

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.     

Diversity in Chemotaxis Mechanisms among the Bacteria and Archaea

The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B. subtilis have been discovered in other organisms. This review emphasizes those used by B. subtilis and these other organisms but also gives an account of the mechanism in E. coli.     

PIKfyve Regulation of Endosome-Linked Pathways

The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5) P ₂, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5) P ₂ is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies.     

Microhomology Directs Diverse DNA Break Repair Pathways and Chromosomal Translocations

Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called “microhomology,” yet the genetic pathway(s) responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR) events after a DNA double-strand break (DSB) in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.     

Widely Conserved Signaling Pathways in the Establishment of Cell Polarity

How are the asymmetric distributions of proteins, lipids, and RNAs established and maintained in various cell types? Studies from diverse organisms show that Par proteins, GTPases, kinases, and phosphoinositides participate in conserved signaling pathways to establish and maintain cell polarity.The asymmetric distribution of proteins, lipids, and RNAs is necessary for cell fate determination, differentiation, and specialized cell functions that underlie morphogenesis (St Johnston 2005; Gonczy 2008; Knoblich 2008; Macara and Mili 2008; Martin-Belmonte and Mostov 2008). A fundamental question is how this asymmetric distribution is established and maintained in different types of cells and tissues. The formation of a specialized apical surface on an epithelial cell seems quite different from the specification of axons versus dendrites in a neuron, or the asymmetric division of a nematode zygote. Yet, remarkably, a conserved molecular toolbox is used throughout the metazoa to establish and maintain cell polarity in these and many other contexts. This toolbox consists of proteins that are components of signal transduction pathways (Goldstein and Macara 2007; Assemat et al. 2008; Yamanaka and Ohno 2008). However, our understanding of these pathways, and their intersection with other signaling networks, remains incomplete. Moreover, the regulation and cross talk between the polarity proteins and other signaling components varies from one context to another, which complicates the task of dissecting polarity protein function. Nonetheless, rapid progress is being made in our understanding of polarity signaling, which is outlined in this article, with an emphasis on the Par proteins, because these proteins play major roles integrating diverse signals that regulate cell polarity (Fig. 1) (see Munro and Bowerman 2009; Prehoda 2009; Nelson 2009).Open in a separate windowFigure 1.An overview of Par complex signaling, showing inputs (bottom) and outputs (top) with cellular functions that are targeted by these pathways (italics).     

新型胞质DNA感受通路：cGAS-STING的研究进展

细胞胞质中存在的游离DNA一直被宿主的固有免疫系统当做潜在的危险信号,但免疫系统识别和清除这些危险信号的机制还不明确.近几年有研究发现,DNA感受器(DNA sensor)是宿主感受DNA和免疫防御的桥梁,目前已经有超过10种DNA感受器被发现,而干扰素刺激基因(stimulator of interferon genes,STING,也称为TMEM173、MPYS、MITA和ERIS)作为一种DNA感受通路下游关键的接头分子,在感受胞质DNA和免疫防御方面起着重要的信号传递作用,胞质中的DNA可通过DNA感受器激活STING,再激活Ⅰ型干扰素和其他细胞因子,进而启动机体的免疫反应.近些年,胞质中游离DNA如何激活STING,进而如何在体内启动免疫反应以产生抗病毒或抗菌作用的机制研究取得了显著进展.最近,在哺乳动物细胞中发现了一种新型的核酸转移酶cGAS(cyclic GMP-AMPsynthase),它能识别DNA并能产生一种内源性的环化二核苷酸cGAMP(cyclic GMP-AMP)激活STING.本文将最近关于cGAS的发现、胞质DNA通过cGAS激活STING及STING活化后激活机体免疫反应的机制进行了综述.