Dictyostelium discoideum chemotaxis: Threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3′,5′-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10⁻³ nM/μm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10⁻¹ nM/μm. In very steep gradients, above 10 nM/μm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum

Chemotaxis, or cell migration guided by chemical cues, is critical for a multitude of biological processes in a diverse array of organisms. Dictyostelium discoideum amoebae rely on chemotaxis to find food and to survive starvation conditions, and we have taken advantage of this system to study the molecular regulation of this vital cell behavior. Previous work has identified phosphoinositide signaling as one mechanism which may contribute to directional sensing and actin polymerization during chemotaxis; a mechanism which is conserved in mammalian neutrophils. In this review, we will discuss recent data on genes and pathways governing directional sensing and actin polymerization, with a particular emphasis on contributions from our laboratory.     

gbpC和gbpD基因在盘基网柄菌细胞趋化性和趋电性运动中的差异研究

目的 趋化性和趋电性是细胞定向迁移的主要方式，并在生物有机体的生理和病理过程中发挥重要作用，但二者存在差异。本文对盘基网柄菌gbpC和gbpD基因在细胞趋电性和趋化性中的作用进行对比研究，以寻找两种迁移方式之间的新差异。方法 将gbpC基因突变株gefT-和gbpD基因突变株gefU-分别置于场强为12 V/cm的直流电场中，分析细胞在电场中的运动方向及运动速度，探讨细胞的趋电性变化；利用电穿孔技术将标记F-actin的Lifeact-GFP质粒转化进入细胞，用荧光显微镜观察活细胞运动时F-actin的分布；用蛋白质印迹技术定量分析细胞的肌球蛋白调节轻链（RLC）在受直流电场刺激后的磷酸化变化情况。结果 gefT-突变株细胞极化消失，但保持与野生型类似的趋电性；gefU-突变株细胞发生超极化，但趋电性显著降低。在直流电场中，突变株细胞和野生型细胞的F-actin主要分布在伪足部位。在电场作用下，细胞株的肌球蛋白RLC磷酸化变化情况存在差异，即野生型细胞以时间依赖的方式发生磷酸化，gefT-突变株细胞先急剧下降，然后再上升，gefU-突变株细胞却以时间依赖方式脱磷酸化。结论 本研究表明gbpC和gbpD基因在盘基网柄菌趋化性和趋电性中的作用不同，暗示了电信号与化学信号确实通过不同的机理指导细胞的定向迁移。     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

细胞色素c在盘基网柄菌细胞凋亡中的作用

细胞色素c在细胞凋亡中发挥着重要的作用,其作用机理在高等真核生物及低等真核生物酵母中已经比较清楚,但在盘基网柄菌(Dictyostelium discoideum)中的作用却没有相关报道.所以我们用western blot和实时荧光定量PCR的方法分别测定了盘基网柄菌前柄细胞和前孢子细胞中细胞色素c的含量及表达量的变化...     

阿拉伯海假交替单胞菌N1230-9两个甲基受体趋化蛋白的功能鉴定

【目的】作为海洋中的特有及优势种群,假交替单胞菌(Pseudoalteromonas)普遍拥有多个甲基受体趋化蛋白(methyl-accepting chemotaxis protein, MCP),探究这些趋化受体的功能。【方法】以太平洋表层海水来源的一株阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9为研究对象,利用软琼脂平板法测试该菌株对23种碳源的趋化能力,继而利用同源重组策略构建2个含sCache结构域MCP编码基因(woc28264和woc27036)缺失突变体,并分析突变体对10种碳源的趋化能力。【结果】菌株N1230-9对海藻糖、麦芽糖、蔗糖、N-乙酰氨基葡萄糖、l-苹果酸、乙酸钠、丙酸钠、丙酮酸钠、柠檬酸和琥珀酸10种碳源具有趋化能力。WOC28264是l-苹果酸和蔗糖的特异性趋化受体,WOC27036则是柠檬酸和琥珀酸的特异性趋化受体。此外,WOC28264和WOC27036还均是N-乙酰氨基葡萄糖和海藻糖的趋化受体。【结论】WOC28264和WOC27036存在重叠的碳源效应物。     

磷脂酰肌醇3激酶通过Akt和ERK抑制盘基网柄菌细胞的趋电性

目的 磷脂酰肌醇3激酶（PI3Ks）通过调控肌动蛋白在细胞定向运动中发挥重要作用。然而,PI3Ks的结构和功能很复杂,人们对PI3Ks在细胞趋电性运动中的作用并不完全清楚。因此,本文以模式生物盘基网柄菌细胞为实验材料,探究其中的PI3K1和PI3K2在细胞趋电性运动中的作用。方法 首先利用CRISPR/Cas9系统介导分别构建PI3K1编码基因pikA基因敲除突变株和PI3K2编码基因pikB基因敲除突变株;随后将2个突变株置于强度为12 V/cm的直流电场中,记录并分析两个突变株的趋电性。结果 数据分析显示,野生型细胞在直流电场中的方向指数为（0.86±0.03）,而pikA-和pikB-突变株在直流电场中的运动方向指数分别为（0.95±0.02）和（0.94±0.03）;此外,野生型细胞在电场中的平均轨迹速度（3.34±0.08）μm/min,而pikA-和pikB-突变株的平均轨迹速度分别为（4.85±0.20）μm/min和（5.48±0.15）μm/min,t检验表明突变株和野生型的方向性指数和运动速度都存在极显著的差异。蛋白质印迹实验结果显示,pikA-和pikB-突变株中...     

Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid

Summary In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase^– mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura ^– phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.     

A model for cell type localization in the migrating slug of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.     

CBP1 Associates with the Dictyostelium Cytoskeleton and Is Important for Normal Cell Aggregation under Certain Developmental Conditions

In cells of the eukaryotic microorganism Dictyostelium discoideum, at least eight small, four-EF-hand Ca²⁺-binding proteins of unknown function are expressed at specific times during development. One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development. To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins. Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1α. Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca²⁺, but the interaction of CBP1 with EF-1α was increased substantially by Ca²⁺. CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca²⁺-dependent. cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular. This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant cAMP. Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation.     

Social insects and social amoebae

The evolution of social groupings in insects, especially wasps, is compared to that of social amoebae (cellular slime moulds). They both show a gamut of colony sizes, from solitary forms to complex colonies with a division of labour. The various ideas as to how there might have been an evolution of complexity within insect societies, such as the role of genetic relatedness, the role of mutualism, the origin of sterility, the manipulation and exploitation of some individuals by others within a colony, are discussed, and then applied to social amoebae. The result is both interesting and instructive: despite some differences, there are many striking parallels, which suggests that there are some common denominators in the formation and evolution of a social existence among organisms.     

Global/temporal gene expression analysis of Escherichia coli in the early stages of symbiotic relationship development with the cellular slime mold Dictyostelium discoideum

Escherichia coli and the cellular slime mold Dictyostelium discoideum form stable viscous symbiotic colonies in the laboratory. To examine changes in E. coli gene expression during establishment of this symbiotic relationship, cells of symbiotic co-cultures and monocultures at various time points were subjected to microarrays analysis. Genes changed significantly over time compared to the initial gene expression level were determined as characteristics of GO function categories. The categories that appeared significantly at the same sampling time points between the two cultures were also identified. Up-regulation of genes from several GO categories associated with polysaccharide synthesis, cell wall degradation, and iron acquisition as well as down-regulation of genes from GO categories associated with biosynthesis through starvation response were observed in co-cultures, indicating exchange of molecules between the two organisms. Up-regulation of genes from several GO categories associated with anaerobic respiration and flagella biosynthesis were also observed, indicating that the environment inside symbiotic colonies was similar to that in developed biofilms. Up-regulation of genes associated with energy-generating systems indicated that E. coli prolonged survival within the symbiotic colony. Thus, E. coli showed not only molecule exchange but also altered expression of various genes in symbiosis with D. discoideum.     

Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans北大核心CSCD

［Objective］ Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata.Due to its ability to grow and fix nitrogen under three conditions, A.caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses.Chemotaxis appears crucial for the growth of A.caulinodansin complicated environment and the construction of associative relationship with the plant.However, little is known about the chemotactic pathway of A.caulinodans.Thus, our study aimed to compare the chemotaxis-like genes of A.caulinodans with those of well-studied species.［Methods］ NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A.caulinodans.HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein （MCP）.［Results］ There was a major chemotaxis cluster in A.caulinodans and the CheR methylated MCPs independently of pentapeptide motif.There were 43 MCP homologs containing diverse signal-sensing architectures in A.caulinodans.In addition,cytoplasmic domains of these MCPs were all composed of 38 heptad repeats.［Conclusion］ Despite the extremely high homology presented between the chemotactic system of A.caulinodans and those of well-studied species, A.caulinodans shows its own unique characteristics.The classification of these chemotactic pathways by comparative genomics enables us to better understand how A.caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

Pitfalls in the measurement of protein carboxyl methylation during chemotaxis of Dictyostelium discoideum

In Dictyostelium discoideum, a chemoacttractant-stimulated incorporation of radioactivity from [methyl-³H]methionine into protein in the presence of cycloheximide has previously been assumed to represent carboxyl methylation. In this paper, however, evidence is presented which demonstrates it to be a non-covalent binding of methionine to structural components of the cell. Certain pitfalls in the assay of carboxyl methylation by measurement of methanol production are described, and the assay has been optimized to avoid measurement of other volatile compounds. When carboxyl methylation is measured as the amount of methanol formed from methylated protein, methanol production is near the limit of detection in all current methods of assay. No increase of methanol production could be observed upon a single or repeated stimulation of aggregative cells with their chemoattractant, cyclic AMP. We conclude that carboxyl methylation is either absent in Dictyostelium, or that it involves only a small amount of specific methyl acceptors.     

Behaviour of dictyostelium discoideum amoebae and Escherichia coli grown together in chemostat culture

When Dictyostelium discoideum amoebae and Escherichia coli were grown together in chemostat culture damped oscillations in the popullation densities of the organisms occurred followed by a sudden increase in bacterial numbers and a concommitant decrease in the number of amoebae. After the system had come to equilibrium altering the dilution rate resulted in a monotonic change in the experimental variables to new steady state levels. A square wave increase in the concentration of limiting nutrient in the feed medium during the oscillatory phase of culture produced a sinusoidal response indistinguishable from that prior to the perturbation. The results are more complicated than those predicted by simple models of microbial predator-prey dynamics although they correspond most nearly to models which incorporate saturation kinetics.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...     

Endomelanconiopsis microspora发酵产物乙酸乙酯提取物对人参核盘菌的抑制机理

【背景】人参菌核病是人参的主要病害之一,严重影响人参的产量。【目的】探索白花蒲公英内生菌(Endomelanconiopsis microspora)发酵产物乙酸乙酯提取物对人参核盘菌的抑制机理。【方法】采用人参核盘菌菌丝生长和孢子萌发试验测定抑制效果;采用显微镜观察菌丝形态变化,通过电导率和核酸含量的变化测定细胞膜通透性,通过丙二醛(malondialdehyde,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)和过氧化氢酶(catalase,CAT)活力的变化测定膜脂过氧化程度。【结果】内生菌E. microspora发酵产物乙酸乙酯提取物能显著抑制人参核盘菌菌丝生长,最小抑菌浓度为3.75 mg/mL,培养6 d后抑制率为76.22%。该提取物能显著抑制人参核盘菌孢子萌发,15.00 mg/mL时抑制效果最好,抑制率达90.69%。提取物影响菌丝形态,增加人参核盘菌细胞膜通透性,造成菌丝内含物外渗,7.50 mg/mL处理10 h后电导率和核酸含量分别比对照组增加30.11%和62.85%。同时提取物显著增加人参核盘菌MDA含量和SOD、POD、CAT活力,7.50 mg/mL处理组呈现先上升后下降的变化趋势,并在12 h时达到最高值。【结论】内生菌E. microspora发酵产物乙酸乙酯提取物通过改变人参核盘菌细胞膜通透性,加剧膜脂过氧化,破坏细胞膜完整性,导致细胞内含物流失,显著抑制孢子萌发和菌丝生长。     

Emergent properties and the context objection to reduction

Reductionism is a central issue in the philosophy of biology. One common objection to reduction is that molecular explanation requires reference to higher-level properties, which I refer to as the context objection. I respond to this objection by arguing that a well-articulated notion of a mechanism and what I term mechanism extension enables one to accommodate the context-dependence of biological processes within a reductive explanation. The existence of emergent features in the context could be raised as an objection to the possibility of reduction via this strategy. I argue that this objection can be overcome by showing that there is no tenable argument for the existence of emergent properties that are not susceptible to a reductive explanation.