Proteomic profiling of the extracellular matrix (slime sheath) of Dictyostelium discoideum

Dictyostelium discoideum has historically served as a model system for cell and developmental biology, but recently it has gained increasing attention as a model for the study of human diseases. The extracellular matrix (ECM) of this eukaryotic microbe serves multiple essential functions during development. It not only provides structural integrity to the moving multicellular pseudoplasmodium, or slug, it also provides components that regulate cell motility and differentiation. An LC/MS/MS analysis of slug ECM revealed the presence of a large number of proteins in two wild‐type strains, NC4 and WS380B. GO annotation identified a large number of proteins involved in some form of binding (e.g. protein, polysaccharide, cellulose, carbohydrate, ATP, cAMP, ion, lipid, vitamin), as well as proteins that modulate metabolic processes, cell movement, and multicellular development. In addition, this proteomic analysis identified numerous expected (e.g. EcmA, EcmD, discoidin I, discoidin II), as well as unexpected (e.g. ribosomal and nuclear proteins) components. These topics are discussed in terms of the structure and function of the ECM during the development of this model amoebozoan and their relevance to ongoing biomedical research.     

Genetic structure of a natural population of Dictyostelium discoideum, a cellular slime mould

Dictyostelium discoideum is a eukaryotic microbe feeding on soil bacteria. A first step towards describing the genetic structure of populations of this species was made by examining multiple isolates from a single locale. The isolates were grown clonally and their RFLP patterns compared, using a probe specific for a family of tRNA genes. Thirty-nine types were distinguished in 54 isolates. To determine if genetic exchange occurs among members of the population, an analysis of linkage disequilibrium was performed on the RFLP data. Little disequilibrium was found, implying gene flow in the population. In conflict with this result is the finding that no recombinant progeny were recovered from many attempted crosses between pairs of isolates. The tentative conclusion is that genetic exchange does not in fact occur, and that the observed shuffling of RFLP bands is caused by insertion and excision of transposons known to be associated with the tRNA genes of Dictyostelium.     

Staurosporine induces tyrosine phosphorylation in Dictyostelium discoideum proteins

The treatment of cells with staurosporine results in inhibition and less frequently activation of protein kinases, in a cell-type specific manner. In the social amoeba Dictyostelium discoideum, staurosporine induces marked changes in cell morphology affecting growth and development. Here we describe that incubation of D. discoideum growing or starved cells with staurosporine results in a rapid and unexpected tyrosine phosphorylation on two polypeptides of approximately 64 and approximately 62 kDa. These proteins emerge as novel substrates for tyrosine phosphorylation opening up new perspectives for the study of cell signalling in D. discoideum.     

盘基网柄菌——研究致病机制的宿主模型

盘基网柄菌作为致病菌宿主模型的研究主要有:筛选致病菌株及相应突变菌株毒性;鉴别对致病菌易感性和抗性的突变细胞宿主;宿主细胞的有效标记、已完成的基因组计划以及宿主细胞与致病菌间信号转导通路的相互作用;这些都表明盘基网柄菌是致病机制研究的理想宿主模型。     

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.     

盘基网柄菌DdLIS1蛋白生物信息学分析

LIS1蛋白是一种与人类无脑回疾病以及细胞癌变相关的重要蛋白。对盘基网柄菌DdLIS1进行生物信息学分析,探究盘基网柄菌能否作为研究人类无脑回疾病及细胞癌变机制的模型。现从NCBI中的Genank找到盘基网柄菌DdLIS1的氨基酸序列,随后进行blastp找到模式生物中相似序列,利用理化性分析网站ProtScale、ProtParam分析DdLIS1的理化性质,通过NCBI中的保守结构域库(CDD)分析DdLIS1的保守结构域,使用MEGA6.0并选用邻位连接法构建系统进化树,分别使用PredictProtein、SWISS-MODEL网站预测Dd LIS1蛋白的二级结构、三维结构。结果得出DdLIS1蛋白全长为419,属于亲水性蛋白,有7个保守结构域,属于WD40家族,与人类和小鼠的氨基酸序列相似性为72%。二级结构中β折叠所占比例最高,为49.40%,α螺旋、随机卷曲分别占该蛋白7.16%、43.44%,与三级结构一致。以上结果说明DdLIS1与LIS1高度相似,有助于盘基网柄菌能够作为研究人类无脑回疾病以及细胞癌变机制的模型。     

Rapid antagonistic coevolution between strains of the social amoeba Dictyostelium discoideum

Social groups face a fundamental problem of overcoming selfish individuals capable of destroying cooperation. In the social amoeba Dictyostelium discoideum, there is evidence that some clones (‘cheaters’) contribute disproportionately to the viable spores in a fruiting body while avoiding the dead stalk cell fate. It remains unclear, however, whether this cheating is actually the product of selection. Here, I report the results of an experimental evolution study designed to test whether clones of D. discoideum will evolve resistance to cheating in the laboratory with genetic variation created only through spontaneous mutation. Two strains, one green fluorescent protein (GFP)-labelled and one wild-type, were allowed to grow and develop together before the wild-type strain was removed and replaced with a naïve strain evolving in parallel. Over the course of 10 social generations, the GFP-labelled strain reliably increased its representation in the spores relative to control populations that had never experienced the competitor. This competitive advantage extended to the non-social, vegetative growth portion of the life cycle, but not to pairwise competition with two other strains. These results indicate strong antagonism between strains, mediated by ample mutational variation for cheating and also suggest that arms races between strains in the wild may be common.     

Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

Phototaxis genes on linkage group V in Dictyostelium discoideum

Abstract During the course of mapping and complementation analysis of phototaxis ( pho ) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK . These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.     

Protein synthesis in cell-free extracts of Dictyostelium discoideum

We have developed an in vitro translation system for the lower eukaryote Dictyostelium discoideum. Active extracts using endogenous mRNA support protein synthesis with optimal Mg²⁺ and K⁺ concentrations of 5 mM and 120 mM, respectively. [³⁵S]Methionine incorporation is linear for more than 2 h. Polypeptides synthesized from endogenous mRNA have sizes ranging from less than 20 to over 100 kDa. Heat-shock proteins are synthesized in vitro in extracts prepared from heat-shocked cells. Possible uses of this system for study of translational control during growth and differentiation are discussed.     

Coh A,a mutation affecting the post aggregative related (par) cohesive system of Dictyostelium discoideum

Three stage-specific cohesive systems operate in D. discoideum: VEG, elaborated by vegetative cells: AR, by aggregation competent cells; and PAR, by post aggregation stage cells. Previous study of a mutant strain JC-5 had shown the stability of its PAR system (but not the AR) to be temperature sensitive. However, the phenotypic expression of this mutation termed Coh A is complicated by the presence in that strain of a preexisting mutant gene Rde A, which accelerates developmental events generally and alters the pattern of morphogenesis. Genetic evidence presented here indicates that the two mutations have been separated by parasexual recombination yielding a Coh A, Rde A⁺ segregant class of which strain JC-36 is a prototype. At the permissive temperature, JC-36 follows a morphogenetic sequence like that of the wild type in respect to timing, morphogenetic pattern, and spore appearance. At the restrictive temperature, it forms normal aggregates at the usual time but exhibits two morphogenetic aberrancies during post aggregative development. First, fruit construction is arrested at a stage approximating the 16 hr “Bottle” stage of the wild type, though more squat and blunt tipped, and then the aggregate regresses. Cytodifferentiation into spores and stalk cells is also blocked. Second, a shift of slugs migrating normally at the permissive temperature to the restrictive causes the latter to disintegrate progressively as they leave clumps of cells behind them within the flattened sheath. JC-36 cells developing at the restrictive temperature also exhibited a decrease in EDTA resistant cohesivity attributable on two grounds to the sensitivity of the PAR system. In addition, the disappearance of the AR system completed in the wild type by the Mexicanhat (18–19 hr) stage is indefinitely arrested at an intermediate level in JC-36.     

The nysB sunD double mutant of Dictyostelium discoideum is blocked in the acquisition of non-degradative resistance to the pea phytoalexin pisatin

Dictyostelium discoideum amoebae can be induced to acquire a non-degradative resistance to the pea phytoalexin pisatin. The nysB sunD double mutant is blocked in the acquisition of this resistance. Using parasexual genetics it has been shown that sunD is a recessive mutation linked to nysB on linkage group VI.     

Responses of Dictyostelium discoideum to Multiple Environmental Stimuli

The movement responses of the cellular slime mold Dictyostelium discoideum to multiple stimuli were investigated. The responses were found to differ depending on the developmental stage of the organism. A novel response, positive gravitaxis, was found in Dictyostelium slugs but not in amoebae. In the presence of a simultaneous light stimulus, gravitaxis is effective only at low fluence rates. Slugs showed positive thermotaxis in a thermal gradient (0.2 °C cm^?1) and ignored the simultaneous light stimulus at low fluence rates (< 10^?3 W m^?2), while at higher fluence rates they moved toward the light source. With a combination of a thermal gradient and gravity Dictyostelium slugs clearly oriented thermotactically ignoring the gravistimulus.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。     

Self- and non-self-recognition in bisexual mating of Dictyostelium discoideum

Cell recognition plays a central part in the sexual process. Although cell-surface molecules involved in gamete recognition have been identified in several organisms, our knowledge of the molecular basis of sexual cell recognition is still limited. We have been studying molecular mechanisms of sexual cell fusion using the lower eukaryote Dictyostelium discoideum . There are homothallic, heterothallic, bisexual and asexual strains in D. discoideum , and how they distinguish between each other to find out proper partners is an interesting and important question. However, analytical studies of sexuality in D. discoideum have been carried out mostly on heterothallic strains, and the polymorphism of the mating system has not yet been thoroughly investigated. In the present study, we extended our analysis to the bisexual mating phenomenon paying special attention to the mechanism of self-incompatibility. We showed that a bisexual strain WS2162 was self-incompatible at the step of sexual cell fusion. Results of antibody inhibition of cell fusion and detection of gp138, a cell-fusion-related protein found in heterothallic strains, suggest that a molecular basis for bisexual and heterothallic mating are common. We propose two models to clarify the mechanisms of self- and non-self discrimination in bisexual mating patterns of D. discoideum .     

Pheromones do regulate sexual development in Dictyostelium discoideum

Based on negative data, Douglas et al refuted earlier work by the O’Day laboratory on the role of pheromones in the sexual development of Dictyostelium discoideum. This letter addresses some of the issues with their study.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.