Distribution of alkaline phosphatase in vegetative dictyostelium cells in relation to the contractile vacuole complex

The structure of the contractile vacuole complex of Dictyostelium discoideum has long been a subject of controversy. A model that originated from the work of John Heuser and colleagues described this osmoregulatory organelle as an interconnected array of tubules and cisternae the membranes of which are densely populated with vacuolar proton pumps. A conflicting model described this same organelle as bipartite, consisting of a pump-rich spongiome and a pump-free bladder, the latter membranes being identified by their alkaline phosphatase activity. In the present study we have employed an antiserum specific for Dictyostelium alkaline phosphatase to examine the distribution of this enzyme in vegetative cells. The antiserum labels puncta, probably vesicles, that lie at or near the plasma membrane and are sometimes, but only rarely, enriched near contractile vacuole membranes. We conclude that alkaline phosphatase is not a suitable marker for contractile vacuole membranes. We discuss these results in relation to the two models of contractile vacuole structure and suggest that all data are consistent with the first model.     

Analysis of 5' nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.     

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。     

聚氨酯泡沫半固定化培养盘基网柄菌

研究了聚氨酯泡沫应用于固定化盘基网柄菌的可行性,发现以简单处理过的聚氨酯泡沫为载体,能够高效实现盘基网柄菌的固定化培养。考察了载体粒径大小、载体量和摇床转速等对固定化培养的影响,在优化的培养条件和固定化条件下,盘基网柄菌的最大细胞密度是悬浮培养的2~4倍。     

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。     

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.     

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.     

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.     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.     

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 .     

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.     

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.     

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.     

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。     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

Induction of Heterothallic and Homothallic Zygotes in Dictyostelium discoideum by Ethylene

Dictyostelium mucoroides -7 (Dm7) and a mutant (MF1) derived from it exhibit homothallic macrocyst formation in the sexual process. As previously shown, the zygote formation during macrocyst formation is induced by a potent plant hormone, ethylene. The present work was undertaken to know if ethylene is also involved in heterothallic and homothallic macrocyst formation in D. discoideum. In heterothallic macrocyst formation between NC4 and V12M2 cells, ethionine, an analogue of methionine, inhibits macrocyst formation through arresting specifically the acquisition process of fusion competence. Such an inhibitory effect of ethionine was almost completely cancelled by co-application of ACC (1-aminocyclopropane-1-carboxylic acid), the immediate precursor of ethylene. Essentially the same effects of ethionine and ACC were also noticed on homothallic macrocyst formation in D. discoideum AC4. Thus it seems most likely that ethylene is required for the acquisition of fusion competence during macrocyst formation, and that in a variety of strains examined there is a common mechanism regulated by ethylene, beyond the difference of sexual modes.     

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.