盘基网柄菌细胞的粘附分子

盘基网柄菌（Dictyostelium discoideum)依赖4种类型细胞粘附系统的表达使其多细胞发育顺利进行。在发育初期,由钙结合蛋白DdCAD-1调节EDTA／EGTA敏感的粘着位点。在发育的多细胞聚集阶段,出现EDTA抗性的粘着位点,由分子是80kD蛋白（gp80)通过同嗜性粘着的相互作用末调节细胞的粘着,它的细胞结合位点是一个八肽序列。由分子量150kD蛋白（gp150)通过异嗜性粘着的相互作用来调节多细胞后聚集的细胞粘着。本文详细讨论了gp80和gp150调节细胞粘着的机制。     

盘基网柄菌(Dictyostelium discoideum)细胞的分化及其调控

本文综述了盘基网柄菌(Dictyostelium dis-coideum)发育过程中细胞类型的诱导和分化,细胞外cAMP及其四种位于细胞表面的受体及PKA(蛋白激酶A)、GSK-3(糖原合成酶激酶)和STATa等在网柄菌发育过程中的作用。     

盘基网柄菌发育中的细胞粘附分子及其信号转导

在盘基网柄菌发育早期,DdCAD-1和csA调节了变形虫细胞间的粘着,调控该过程的机制类似于胚胎发育中上皮细胞层的闭合。完成网柄菌发育的一个必需分子是gpl50异嗜性粘附分子。盘基网柄菌β-连环蛋白同源物Aardvark(Aar)的缺乏使细胞间失去粘着连接,Aar也有信号转导功能,调控了前孢子细胞基因的表达。因此,细胞间的粘着是盘基网柄菌发育的一个重要组成部分,并与调控形态发生过程的信号转导有密切相互作用关系。     

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。     

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...     

cAMP受体在盘基网柄菌发育中的作用

在盘基网柄菌(Dictyostelium discoideum)发育的过程中,环腺苷酸(cAMP)的4种受体(cAR1～4)发挥极为重要的作用,它们能将胞外不同浓度的cAMP所代表的信息传递到胞内,调控发育。cAR1和cAR3作用相似,但各有侧重：cAR1主要在发育前期参与cAMP脉冲的形成,控制细胞爬行的方向,开启多细胞发育的过程;而cAR3主要在细胞分化时期调节GSK3等信号分子。cAR2／4两作用相似,但表达的时间和空间不同,在不同区域调控细胞分化及参与多细胞体的形态建成。     

盘基网柄菌分化逆转引起mRNA的快速降解及抗菌素对其影响

诺加拉霉素能有效封锁盘基网柄菌转录,放线菌酮和嘌呤霉素对抑制该菌蛋白合成有相近效力。盘基网柄菌分化发育后期积累的mRNAs,在细胞被分散(分化逆转)时,专一地被快速降解。诺加拉霉素、放线菌素D和柔红霉素合用稳定某些快速降解的mRNAs。放线菌酮稳定所有mRNAs。嘌呤霉素不能稳定后期mRNAs,且有促进降解作用。表明后期mRNAs的快速降解不需要新蛋白合成,分化逆转只增强了已有酶的作用。     

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。     

盘基网柄菌细胞发育过程中尿囊酸酶的亚细胞定位

利用胶体金免疫电镜技术,观察了盘基网柄菌细胞分化与凋亡过程中胞内尿囊酸酶的位置变化。结果表明,在细胞聚集期细胞产生的尿囊酸酶主要分布于线粒体及周围细胞质内。到了细胞丘时期,尿囊酸酶只特异地存在于发生内自噬的线粒体内,且仅局限于线粒体因内自噬产生的空泡区域,这些发生线粒体内自噬的细胞将分化成前孢子细胞。随着前孢子细胞分化的进行,尿囊酸酶颗粒在细胞内分布逐渐减少,在靠近质膜处的空泡内还能观察到一些酶颗粒;而另一些细胞内,几乎所有的胞器内都能观察到酶颗粒,一直延续至柄细胞形成。从中可以看到尿囊酸酶在将发育成孢子细胞和柄细胞两种类型细胞内的分布位置明显不同,结果提示了尿囊酸酶蛋白与盘基网柄菌细胞分化和凋亡调控途径有密切关系。     

Signalling molecules involved in cellular differentiation during Dictyostelium morphogenesis

GSK-3, Dd-STATa, PKA, rZIP and Ras all play important roles in cell type determination of Dictyostelium discoideum. The fact that homologs of these proteins also function in metazoan development emphasizes the importance of Dictyostelium as a model microbial organism for studying the molecular mechanisms that regulate development. The recent elaboration of the central role for GSK-3 in cell type determination has been of particular importance. The stimulatory effect of extracellular cAMP on GSK-3 activity has been shown to act through the cell surface receptor cAR3 and a tyrosine protein kinase ZAK1, which directly activates and phosphorylates GSK-3. Several proteins, including Dd-STATa, have been identified as substrates for GSK-3, and are therefore potential transducers of the signals involved in cell type determination.     

A gene encoding, prespore-cell-inducing factor in Dictyostelium discoideum

Two factors that exist in conditioned medium (CM) of Dictyostelium discoideum induce amoebae to differentiate into prespore cells when they are incubated at a very low cell density in submerged monolayer culture. Previously, we purified one of them, a glycoprotein factor with an apparent molecular mass of 106 kDa, and we named it psi factor (psi, prespore-inducing factor). Based on the partial amino acid sequence of the purified psi factor, we have isolated the corresponding cDNA clone, which is expressed maximally at the loose mound stage. The cDNA encodes a novel protein and the predicted molecular mass of the mature secreted protein is 60 kDa. Knockout mutant strains of the psi factor gene, psiA(-), were created by targeted integration. Although these mutant strains appear to develop normally, CM from these mutants showed reduced prespore-cell-inducing activity. Rescuing the mutant strains by expression of psi factor under control of a constitutive promoter causes overproduction of psi factor protein and CM from such cells showed a 20-fold higher level of prespore-cell-inducing activity than that from wild-type cells. Further, CM from parental cells induced prespore cell division, while that from psiA null strains showed no cell division inducing activity. Our results indicate that psi factor protein is a novel type of growth factor that does not belong to any of the families of growth factor so far identified in animals.     

Zinc ions promote prestalk-to-stalk and prespore-to-stalk conversions in Dictyostelium discoideum

Abstract To clarify the mechanism of stalk cell differentiation in Dictyostelium discoideum (strain NC4), we have examined the effects of Zn²⁺ on in vitro cell differentiation of prestalk and prespore cells isolated from normally formed slugs. Prestalk cells did not differentiate into stalk cells under submerged conditions, but in the presence of the stalk-inducing factor-1 (DIF-1) at 100 nM or Zn²⁺ at 5 mM, a small number of the cells (< 15%) differentiated into stalk cells. Interestingly, Zn²⁺ in combination with DIF-1 induced the prestalk-to-stalk conversion at high efficiencies (approx. 60%). Furthermore, isolated prespore cells were also converted to stalk cells at high efficiencies (approx. 50%) in the presence of both DIF-1 and Zn²⁺, while the conversion poorly occurred in the absence of Zn²⁺. These results indicate that Zn²⁺ may mimic some cellular interaction(s) which are required for stalk cell formation in this strain.     

Differentiation inducing factors in Dictyostelium discoideum: A novel low molecular factor functions at an early stage(s) of differentiation

There are reports that secreted factor(s) are involved in prespore cell differentiation in Dictyostelium discoideum, but the structures and functions of the various factors have not been elucidated. Previously, we described two prespore cell‐inducing factors in conditioned medium; one was a glycoprotein named prespore cell‐inducing factor (ψ factor, or PSI‐1), and the other, a heat stable dialyzable factor(s). In the present paper, we purified and characterized the most potent prespore cell‐inducing activity in dialysates. The factor began to be secreted after the onset of starvation and stopped being secreted once the cells had aggregated, which was earlier than the onset of the ψ factor gene expression. In addition, unlike ψ factor, its secretion did not appear to depend on activation of protein kinase A. Interestingly, the purified factor not only induced prespore cell specific genes such as pspA and cotC but also a prestalk‐cell specific gene, ecmB in vitro. The purified factor is tentatively designated polyketide‐like factor (PLF), because it seems to be a novel polyketide with 208 Da. Half maximal induction of prespore cell was obtained with 26 nmol/L of PLF. We propose that PLF plays a key role in the acquisition of differentiation commitment, before the ψ factor induces specifically prespore cell differentiation.     

Altered cell-type proportioning in Dictyostelium lacking adenosine monophosphate deaminase.

The proportions of prespore and prestalk cells in Dictyostelium discoideum are regulated so that they are size invariant and can adjust when the ratio is perturbed. We have found that disruption of the gene amdA that encodes AMP deaminase results in a significantly increased proportion of prestalk cells. Strains lacking AMP deaminase form short, thick stalks and glassy sori with less than 5% the normal number of spores. The levels of prestalk-specific mRNAs in amdA(-) cells are more than twice as high as those in wild-type strains and prespore-specific mRNAs are reduced. Using an ecmA::lacZ construct to mark prestalk cells, we found that amdA(-) null slugs have twice the normal number of prestalk cells. The number of cells expressing an ecmO::lacZ construct was not affected by loss of AmdA, indicating that the mutation results in an increase in PST-A prestalk cells rather than PST-O cells. This alteration in cell-type proportioning is a cell-autonomous consequence of the loss of AMP deaminase since mutant cells developed together with wild-type cells still produced excess prestalk cells and wild-type cells carrying the ecmA::lacZ construct formed normal numbers of prestalk cells when developed together with an equal number of amdA(-) mutant cells.     

Calcium levels during cell cycle correlate with cell fate of Dictyostelium discoideum

Levels of intracellular calcium, (Ca(2+))(i), from different stages of cell cycle of Dictyostelium discoideum were monitored using the fluorescent Ca(2+)-sensitive dye, Indo 1. Combinations of Ca(2+)-ionophore (A23187) and Ca(2+)-chelator (EGTA) resulted in the inhibition of progression of cell cycle. This delay was caused due to block in G(2)/M-->S phase transition of the cell cycle. Rescue of the cell cycle progression was made with 0.5 m m of exogenous Ca(2+). High (Ca(2+))(i)levels overlapped with the S-phase, of the cell cycle.Results indicate that a high (Ca(2+))(i)level during S-phase is not required for cell cycle progression but for cell-type choice mechanism at the onset of starvation, and these cells tend to follow the prestalk pathway.     

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.     

Mutation of the Dictyostelium fbxA gene affects cell-fate decisions and spatial patterning

Cell-fate decisions and spatial patterning in Dictyostelium are regulated by a number of genes. Our studies have implicated a gene called fbxA, which codes for an F-box protein, in these pathways. The FbxA protein is one of the controls on a cAMP phosphodiesterase called RegA, mediating its degradation via ubiquitin-linked proteolysis. Using marked strains, we showed that the fbxA^– mutant has defective cell-type proportioning, with a dearth of prestalk cells compared to prespore cells. In this work, we show that this effect occurs earlier during the 24 hour developmental cycle than previously thought. The normal sorting of the prestalk and prespore cells in aggregates and mounds is not affected by the mutation. The mutant cells sort abnormally at the tipped mound stage, when prespore and prestalk cells normally distribute into their proper compartments. The fbxA^– mutant forms prestalk cells in low numbers when not in chimeras, but in the presence of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form non-viable stalk cells. In an attempt to identify the signal transduction pathway that mediates proportionality in prestalk and prespore cells, we asked whether certain signal transduction mutants were immune to the effects of the fbxA^–cells and formed spores in chimeras.     

Induction of Cell Differentiation of Isolated Cells in Dictyostelium discoideum by Low Extracellular pH

In Dictyostelium discoideum , the formation of multicellular masses is necessary for cell differentiation. However, the present study shows that amoebae of strain V12M2 efficiently differentiate to prespore or stalk cells under submerged incubation in a simple medium containing cAMP and salts without cell contact, only if the pH of the medium is maintained at acidic values; differentiation scarcely occurs in the neutral pH range. The optimum pH values for prespore and stalk cell differentiation are 5.1 and 4.5, respectively. In addition to the extracellular pH, Mg ions and the concentration of cAMP also affect the choice of the differentiation pathway. The time courses of differentiation of both cell types under optimum conditions are also presented.