盘基网柄菌中分化诱导因子信号与细胞命运决定

对盘基网柄菌发育过程中分化诱导因子(DIF)的作用及其机制进行了综述,包括DIF对盘基网柄菌前柄细胞、柄细胞分化的作用以及DIF的生物合成、DIF的诱导、降解失活、DIF对细胞命运和细胞比例的调节及其作用机制等。     

盘基网柄菌细胞黏附分子cadA/DdCAD-1在细胞分化及细胞命运决定中的作用

盘基网柄菌细胞黏附分子DdCAD-1是在细胞发育过程中最先表达的黏附分子,为了研究DdCAD-1在盘基网柄菌细胞发育中的作用,将cadA基因的突变株cadA-细胞用中性红染料染色,发育成的蛞蝓体显示cadA-细胞的前柄细胞/前孢子细胞的分化出现明显的障碍,外源表达的重组蛋白His6-DdCAD-1与cadA-细胞作用一段时间后,这种现象得到了改善。另外,cadA-细胞的孢子产率也有所降低,外源重组蛋白也可以拯救该表现型。表达DdCAD-1的细胞与cadA-细胞共同发育所形成的嵌合体显示表达DdCAD-1的细胞占据在拔顶期结构的顶端及尾部,而这些结构都在非孢子区,最终会死亡。提示DdCAD-1对于细胞分化及细胞命运决定有重要意义。     

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。     

PKA在盘基网柄菌(Dictyostelium discoideum)多细胞发育中的作用

在盘基网柄菌(Dictyosteliumdiscoideum)多细胞发育中,蛋白激酶A(proteinkinaseA,PKA)发挥多重作用.细胞聚集阶段,PKA调节腺苷酰环化酶的活性,中转cAMP,诱导dut、pdi等一些发育早期的基因表达;参与启动聚集后的细胞分化和形态构成,增强GBF活性,激活前孢子细胞特有基因的表达;它还精密调控前柄细胞特有基因ecmB的表达,准确启动拔顶发育,诱导孢柄和孢子的成熟.子实体形成后,PKA又是维持孢子休眠和保证孢子有效萌发的必需因子.在PKA调控下,盘基网柄菌有条不紊地完成整个发育过程.     

盘基网柄菌发育机制研究进展

盘基网柄菌细胞与哺乳类细胞在行为、结构和信号通路方面,有很多相同或相似之处。用其作为研究发育机制的模型,为解决哺乳类发育中的一些难题可以发挥重要作用。该文详细讨论了分化诱导因子DIF-1、环磷酸腺苷c AMP、转录因子Srf A和Stk A、粘附分子gp150在盘基网柄菌发育过程中的作用及机制。关于DIF-1、c AMP和gp150信号通路间相互关系还缺乏系统研究,值得进一步深入研究。对这类问题进行深入研究可能有助于阐明多细胞动物起源及演化机制。     

gp150蛋白在盘基网柄菌发育中的作用及粘附分子间关系的分析

饥饿的盘基网柄菌进入多细胞发育期 ,在发育早期 ,AK12 7细胞 (gp150突变细胞 )能表达DdCAD 1和 gp80两种粘附分子 ,但它们不足以促进细胞继续发育 ,发育停留在细胞疏松结合阶段。粘附分子 gp150调节的细胞与细胞间的粘着影响了细胞丘“突出”的形成 ,由此影响了盘基网柄菌多细胞发育的形态发生。TL93细胞 (DdCAD 1和gp80突变细胞 )能完成发育。主要原因是在细胞流发育阶段就表达了gp150分子 ,在细胞粘着的功能上有替代DdCAD 1和 gp80的作用。因此 gp150蛋白对盘基网柄菌多细胞发育有着不可或缺的作用     

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。     

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。     

将盘基网柄菌用于本科细胞生物学综合性实验教学初探——电穿孔转化绿色荧光蛋白

盘基网柄菌(Dictyostelium discoideum)是一个应用广泛的模式生物,非常适合用来研究胞质分裂、细胞运动、吞噬作用、趋化性、趋电性、信号转导以及个体发育过程中的细胞分化。该实验主要介绍电穿孔技术转化绿色荧光蛋白标记肌动蛋白基因质粒(Lifeact-GFP)进入盘基网柄菌活细胞中,抗性筛选(潮霉素B)获得阳性克隆子,最后借助荧光显微镜观察绿色荧光蛋白标记的微丝在盘基网柄菌的分布情况。综合性实验训练可提高学生的学习兴趣和综合运用理论知识的能力,进而可培养学生的科学研究思维。     

Prespore cell inducing factor, ψ factor,controls both prestalk and prespore gene expression in Dictyostelium development

Prespore cell‐inducing (psi, ψ) factor (PsiA), encoded by the psiA gene of Dictyostelium, is a secreted signal glycoprotein that induces prespore cell differentiation when added to monolayer cultures. In situ hybridization during normal development showed that the psiA gene is highly expressed in scattered cells at the mound stage and in prespore cells at the onset of culmination. The conventional prespore‐cell marker genes, cotC and pspA, were expressed normally in psiA^? and psiA overexpressing strains. Expressions of rnrB and cudA are repressed in the prestalk cells of a wild type slug to render prespore specific pattern. However, a promoter‐reporter fusion gene, rnrB:lacZ, showed an ectopic expression in the prestalk cells of the psiA^? strain while cudA(psp):lacZ did so in those of the psiA overexpressing strain. Overexpression of psiA delayed expression of the prestalk specific gene, ecmB, during development, while knocking out psiA promoted its expression. In addition, overexpression inhibited DIF‐1‐induced stalk formation in monolayer cultures. Together with the known prespore inducing activity, the results indicate that PsiA regulates both prespore and prestalk/stalk cell differentiation. These results indicate that PsiA is also involved in prestalk cell differentiation.     

Glutathione is required for growth and prespore cell differentiation in Dictyostelium

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.     

A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.

We are studying cell differentiation in Dictyostelium discoideum by examining the regulation of genes that are preferentially expressed in different cell types. A system has been established in which prestalk- and prespore-cell-specific genes are expressed in single cells in response to culture conditions. We confirm our previous results showing that cyclic AMP induces prestalk genes and now show that it is also required for prespore gene induction. The expression of both classes of genes is additionally dependent on the presence of a factor(s) secreted by developing cells which we call conditioned medium factor(s). An assay for conditioned medium factor(s) shows that it is detectable within 2.5 h after the onset of development. Conditioned medium factor(s) also promotes the expression of genes induced early in development, but has no detectable effect on the expression of actin genes and a gene expressed maximally in vegetative cells. In the presence of conditioned medium factor(s), exogenous cyclic AMP at the onset of starvation fails to induce the prespore and prestalk genes. The addition of cyclic AMP between 2 and 12 h of starvation results in rapid prestalk gene expression, whereas prespore genes are induced at an invarient time (approximately 18 h after the onset of starvation). These data suggest that cyclic AMP and conditioned medium factor(s) are sufficient for prestalk gene induction, whereas an additional parameter(s) is involved in the control of prespore gene induction. In contrast to several previous studies, we show that multicellularity is not essential for the expression of either prespore or prestalk genes. These data indicate that prespore and prestalk genes have cell-type-specific as well as shared regulatory factors.     

Opposite effects of adenosine on two types of cAMP-induced gene expression in Dictyostelium indicate the involvement of at least two different intracellular pathways for the transduction of cAMP signals

Adenosine promotes the cAMP-induced increase of mRNAs, probed with the cDNAs D11 and D14, which are preferentially expressed in prestalk cells, while it inhibits cAMP-induced prespore gene expression. Half-maximal inhibition of prespore gene expression occurs at about 300 muM, while prestalk stimulation by adenosine occurs at about 100-fold lower concentrations and requires the presence of cAMP. These results indicate that adenosine interferes with the transduction to cAMP to gene expression and suggest the involvement of two different adenosine target sites. Our data furthermore indicate that the transduction of extracellular cAMP to prespore gene or prestalk gene expression occurs via divergent pathways.     

Lithium respecifies cyclic AMP-induced cell-type specific gene expression in Dictyostelium

We investigated the effect of LiCl on pattern formation and cAMP-regulated gene expression in Dictyostelium discoideum. In intact slugs, 5 mM LiCl induces an almost complete redifferentiation of prespore into prestalk cells. We found that LiCl acts by interfering with the transduction of extracellular cAMP to cell-type-specific gene expression; LiCl inhibits the induction of prespore-specific gene expression by cAMP, while it promotes the induction of prestalk-associated gene expression by cAMP. Our results indicate that two divergent pathways transduce the extracellular cAMP signal to, respectively, prestalk and prespore gene expression.     

Cytochrome c and insect cell apoptosis

Abstract The role of cytochrome c in insect cell apoptosis has drawn considerable attention and has been subject to considerable controversy. In Drosophila, the majority of studies have demonstrated that cytochrome c may not be involved in apoptosis, although there are conflicting reports. Cytochrome c is not released from mitochondria into the cytosol and activation of the initiator caspase Dronc or effector caspase Drice is not associated with cytochrome c during apoptosis in Drosophila SL2 cells or BG2 cells. Cytochrome c failed to induce caspase activation and promote caspase activation in Drosophila cell lysates, but remarkably caused caspase activation in extracts from human cells. Knockdown of cytochrome c does not protect cells from apoptosis and over‐expression of cytochrome c also does not promote apoptosis. Structural analysis has revealed that cytochrome c is not required for Dapaf‐1 complex assembly. In Lepidoptera, the involvement of cytochrome c in apoptosis has been demonstrated by the accumulating evidence. Cytochrome c release from mitochondria into cytosol has been observed in different cell lines such as Spodoptera frugiperda Sf9, Spodoptera litura Sl‐1 and Lymantria dispar LdFB. Silencing of cytochrome c expression significantly affected apoptosis and activation of caspase and the addition of cytochrome c to cell‐free extracts results in caspase activation, suggesting the activation of caspase is dependent on cytochrome c. Although Apaf‐1 has not been identified in Lepidoptera, the inhibitor of apoptosome formation can inhibit apoptosis and caspase activation. Cytochrome c may be exclusively required for Lepidoptera apoptosis.     

A 3-D model used to explore how cell adhesion and stiffness affect cell sorting and movement in multicellular systems

A three-dimensional mathematical model is used to determine the effects of adhesion and cell signalling on cell movements during the aggregation and slug stages of Dictyostelium discoideum (Dd) and to visualize cell sorting. The building blocks of the model are individual deformable ellipsoidal cells, where movement depends on internal parameter state (cell size and stiffness) and on external cues from the neighboring cells, extracellular matrix, and chemical signals. Cell movement and deformation are calculated from equations of motion using the total force acting on each cell, ensuring that forces are balanced. The simulations show that the sorting patterns of prestalk and prespore cells, emerging during the slug stage, depend critically on the type of cell adhesion and not just on chemotactic differences between cells. This occurs because cell size and stiffness can prevent the otherwise faster cells from passing the slower cells. The patterns are distinctively different when the prestalk cells are more or less adhesive than the prespore cells. These simulations suggest that sorting is not solely due to differential chemotaxis, and that differences in both adhesion strength and type between different cell types play a very significant role, both in Dictyostelium and other systems.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.