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。     

盘基网柄菌AK127细胞超微结构的观察

盘基网柄菌AK127细胞是gP150蛋白基因被剔除的突变细胞。为探明发育期间AK127细胞亚显微结构特征,用透射电镜观察了发育14h、16h、20h的细胞,结果表明:发育14h细胞内含丰富内质网系统,由内质网组织围裹细胞质密度明显低于周围的细胞质,能清楚地观察到多层膜组成的多膜结构。细胞核的内核膜产生凹陷,使内外核膜间产生一个含丝状物质的泡状空间,内核膜上可见螺旋状染色物质,外核膜表面布满颗粒状物质。发育到16 h时,多膜结构内某些膜开始解体,形成自噬泡。线粒体膜性结构完整。发育20 h细胞内有一个内含数个多膜结构的大自噬泡。据此笔者推测多膜结构作为一个储备营养成分"仓库",为维持细胞生命所用。这些数据提示gp150分子的缺失对于细胞的结构和生理过程均有较大影响,gp150分子在细胞生长和发育过程中起重要作用。     

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DJ-1蛋白的原核表达及抗体制备

盘基网柄菌(Dictyostelium discoideum)是研究神经退行性病变的模式生物,可用以研究帕金森病相关基因DJ-1的作用和致病机理。本研究设计特异性引物扩增了人类DJ-1的盘基网柄菌同源基因片段DAM,并利用酶切位点Bam HⅠ和HindⅢ将DAM插入质粒载体p QE-30产生重组载体pPROF696,序列测定后对DJ-1进行了生物信息学分析。同时,通过电穿孔法将pPROF696转入大肠杆菌M15,并利用IPTG诱导DJ-1蛋白表达。经SDS-PAGE分析、Western-blot印迹和His标签树脂对DJ-1粗蛋白纯化后进行抗体制备,并在大肠杆菌M15转化株和盘基网柄菌转化株与野生型内对抗体进行检验。结果表明:本研究成功构建了盘基网柄菌DJ-1的原核表达载体pPROF696,所制备的盘基网柄菌DJ-1多克隆抗体能成功检测大肠杆菌M15和盘基网柄菌野生型与转化株中的DJ-1蛋白。这为今后检测盘基网柄菌细胞内DJ-1的表达水平,建立其与盘基网柄菌表现型相关性,标记DJ-1的亚细胞定位和进一步研究DJ-1在帕金森病中的作用机理奠定了基础。     

盘基网柄菌发育期间gp150蛋白与蛋白激酶A相关性的研究

盘基网柄菌进入多细胞发育阶段后,野生型KAx-3细胞的盘基网柄菌蛋白激酶A（DdPKA）活性分别在12,16,20h时显著升高,这一变化趋势与细胞形态学上的分化有关;而突变型AK127细胞（gp150蛋白缺失）的DdPKA活性则一直保持在较高水平,直至22h才缓慢下降。两种细胞类型中24h的DdPKA活性都再一次升高。总体而言,AK127细胞的DdPKA活性要比KAx-3细胞高。这表明AK127细胞可能因缺失了gp150蛋白而导致DdPKA活性调控失去控制。在KAx-3细胞的分化过程中,前柄细胞（prestalk cells,pst）DdPKA的活性在16～18h缓慢上升,但在20h时显著下降;前孢子细胞（prespore cells,psp）中DdPKA的活性在18h时显著下降,但在20h时又迅速恢复,并达到前柄细胞中DdPKA活性的两倍。激光共聚焦结果显示,在KAx-3发育的关键阶段,DdPKA两种亚基的胞内定位并不一致,DdPKA-R亚基在空间位置上更为靠近gp150蛋白,甚至互相重叠。以上结果表明,gp150蛋白可能通过影响DdPKA-R的活性来调控前柄细胞的凋亡和前孢子细胞的分化。     

新型重组糖蛋白表达载体--盘基网柄菌

近年来,盘基网柄菌作为异源重组糖蛋白表达载体的研究受到了学术界的重视,已经有多种具有生物活性的复杂糖蛋白成功地得到了表达。通过对表达产物的研究发现,盘基网柄菌具有各种翻译后加工机制,例如磷酸化、酰基化及形成GPI(糖基磷脂酰基醇)锚点等,具有类似于高等动物的糖基化修饰能力。与哺乳动物细胞表达载体相比较,盘基网柄菌具有培养成本低廉、细胞生长迅速及易于大规模培养的优势。盘基网柄菌有可能发展成为一种有重要应用前景的糖蛋白表达载体系统。     

The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.     

Purification and partial characterization of a cell adhesion molecule (gp150) involved in postaggregation stage cell-cell binding in Dictyostelium discoideum.

A cell surface glycoprotein of apparent Mr 150,000 (gp150) has been implicated in mediating EDTA-resistant cell-cell adhesion in Dictyostelium discoideum. A simple purification scheme making use of high-performance liquid chromatography has been devised to purify gp150 to near homogeneity. Purified gp150 was capable of neutralizing the effect of a rabbit antiserum raised against gel-purified gp150, which was previously reported to be a potent inhibitor of cell-cell adhesion (Geltosky, J. E., Weseman, J., Bakke, A., and Lerner, R. A. (1979) Cell 18, 391-398). The binding of 125I-labeled gp150 to intact cells was both dose-dependent and saturable, demonstrating the presence of specific cell surface binding sites for gp150. When reassociation of postaggregation stage cells was carried out in the presence of soluble gp150, aggregate formation was strongly inhibited. In contrast, gp150 failed to exert any effect on cells at the aggregation stage. The inhibitory effect of gp150 was sensitive to protease treatment, suggesting that the protein moiety is crucial to gp150 function. These results, taken together, provide direct evidence that gp150 is a cell-cell adhesion molecule involved in cell-cell binding in the postaggregation stage of Dictyostelium development.     

Cell-cell adhesion in Dictyostelium discoideum

Three separate mechanisms of cell-cell adhesion have been shown to appear at different stages of development in Dictyostelium discoideum. During the first few hours of development, the cells synthesize and accumulate a glycoprotein of 24,000 daltons (gp24) that is positioned in the membrane. The time of appearance of gp24 correlates exactly with the time of appearance of cell-cell adhesion in two strains in which temporal control varies by several hours. Antibodies specific to gp24 are able to block cell-cell adhesion during the first few hours of development but not during later development. By 8 hr of development, another glycoprotein, gp80, that is not recognized by antibodies to gp24 accumulates on the surface of cells. This membrane protein mediates an independent adhesion mechanism during the aggregation stage that is resistant to 10 mM EDTA. Antibodies specific to gp80 can block EDTA-resistant adhesion during this stage. During subsequent development, gp80 is removed from the cell surface and replaced by another adhesion mechanism that is insensitive to antibodies to either gp24 or gp80. A lambda gt11 expression vector carrying a Dictyostelium cDNA insert was isolated that directs the synthesis of a fusion protein recognized by antibodies specific to gp24. This cDNA was used to probe a genomic library. A clone carrying a 1.4-kb insert of genomic DNA was recognized by the cDNA and shown to hybridize to a 0.7-kb mRNA that accumulates early in development. This unusually small RNA could code for the small protein, gp24. Southern analysis of restriction fragments generated by various enzymes on Dictyostelium DNA with both the cDNA and genomic clones indicated the presence of two tandem copies of the gene. This may account for the failure to recover mutations resulting in the lack of gp24. Mutations have been recovered that result in the lack of accumulation of gp80, and cells carrying these mutations have been shown to be missing the second adhesion mechanism. These mutant strains are able to complete development because the other adhesion mechanisms are not impaired. Sequential addition of adhesion mechanisms provides a means for the formation of multicellular organisms from previously solitary cells.     

Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.     

Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum

A membrane glycoprotein of 24,000 Da (gp24) was purified from developed cells of Dictyostelium discoideum and shown to neutralize a crude antiserum (R695) that blocks EDTA-sensitive cell-cell adhesion during the early developmental stages of this organism. Purified gp24 was used to raise rabbit polyclonal antibodies and mouse monoclonal antibodies. Rabbit antiserum R851 was shown to be highly specific to gp24 by both Western analysis and immunoprecipitation. IgG of R851 is able to block adhesion of dissociated cells swirled in suspension. Adhesion of wild-type cells is blocked by R851 antibodies during the first 8 hr of development but not thereafter when other adhesion mechanisms come into play. The glycoprotein gp80 plays an essential role in the second adhesion system that appears during the aggregation stage of D. discoideum. By adding both anti-gp24 and anti-gp80 antibodies, adhesion of aggregation stage cells could be blocked. Late in development a third adhesion mechanism appears that is not blocked by either antibodies to gp24 or gp80 or both antibodies together. Western analysis and immunoprecipitation with monoclonal antibody mLJ11, specific for gp24, indicated that gp24 is absent in cells growing exponentially on bacteria but is rapidly synthesized and accumulated following the initiation of development. Synthesis of gp24 is maximal during the first 4 hr of development and then continues at a reduced rate throughout the remainder of development. The coordinate appearance of gp24 and EDTA-sensitive cell-cell adhesion as well as the ability of this glycoprotein to neutralize the adhesion blocking activity of R695 and R851 antibodies indicates that it plays a role in early cell-cell adhesion.     

Cell-cell adhesion and morphogenesis in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire EDTA-resistant cell-cell adhesion at the aggregation stage. The EDTA-resistant cell binding activity is associated with a cell surface glycoprotein of Mr 80,000 (gp80), which mediates cell-cell binding via homophilic interaction. Analysis of the structure of gp80 deduced from cDNA sequence reveals the presence of three internally homologous segments in the NH2-terminal domain, which also contains regions with homology to the neural cell adhesion molecule. Secondary structure predictions show an abundance of beta-structures and very few alpha-helices. This is confirmed by circular dichroism measurements. It is likely that the homologous segments are organized into globular structures, extended from the cell surface by a Pro-rich stalk domain. The cell binding activity of gp80 resides within the first globular repeat of the NH2-terminal domain and has been mapped to a 51 amino acid region between Val123 and Leu173. Synthetic oligopeptides corresponding to sequences within this region have been prepared and assayed for their ability to bind to cell surface gp80. Results lead to identification of the homophilic binding site to an octapeptide sequence within this region. Synthetic peptides containing this octapeptide sequence and univalent antibodies directed against this site block the formation of organized cell streams during aggregation. Although cell aggregates are eventually formed, most fail to undergo further development to give rise to slugs and fruiting bodies, indicating that cell-cell adhesion involving gp80 is an important step in normal morphogenesis.     

Inhibition of cell-cell adhesion and morphogenesis of Dictyostelium by carnitine.

Carnitine (gamma-trimethylammonium beta-hydroxy-butyric acid) possesses the novel property of preventing cell aggregation elicited by clusterin or by fibrinogen (I.B. Fritz and K. Burdzy, J. Cell. Physiol., 140:18-28 [1989]). In investigations reported here, we show that carnitine also affects cell-cell adhesion in Dictyostelium discoideum, a cellular slime mold whose cells interact in specific and complex manners during discrete stages of development. Two types of cell adhesion systems sequentially appear on the surface of developing Dictyostelium cells, involving the surface glycoprotein gp24 which mediates EDTA-sensitive binding sites, and the surface glycoprotein gp80 which mediates the EDTA-resistant binding sites. Addition of increasing concentrations of D(+)-carnitine and L(-)-carnitine resulted in a progressive inhibition of both the EDTA-sensitive binding sites and the EDTA-resistant binding sites of Dictyostelium cells at different stages of development. In contrast, comparable or higher concentrations of choline, acetyl-beta-methylcholine, or deoxycarnitine had no detectable effects on cell aggregation. Concentrations of carnitine required for 50% inhibition of EDTA-resistant adhesion sites were found to be dependent upon levels of gp80 expressed by Dictyostelium, with greatest inhibition by carnitine of reassociation of cells containing the lowest levels of gp80. Removal of carnitine from cells by washing resulted in the rapid restoration of the ability of Dictyostelium to form aggregates and to resume normal development. We discuss possible mechanisms by which carnitine inhibits the aggregation of cells.     

Cell-cell adhesion molecules in Dictyostelium

Multicellularity in the cellular slime mold Dictyostelium discoideum is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive adhesion sites are expressed very early in the development cycle and a surface glycoprotein of 24,000 Da is known to be responsible for these sites. The EDTA-resistant contact sites begin to accumulate on the cell surface at the aggregation stage of development. Several glycoproteins have been implicated in the EDTA-resistant type of cell-cell binding and the best characterized one has an Mr of 80,000 (gp80). gp80 mediates cell-cell binding via homophilic interaction and its cell binding site has been mapped to an octapeptide sequence. The mechanism by which gp80 mediates cell-cell adhesion will be discussed.     

The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum

Dictyostelium discoideum expresses a developmentally regulated cell surface glycoprotein of Mr 80,000 (gp80), which has been implicated in the formation of the EDTA-resistant contact sites A at the cell aggregation stage. To determine whether gp80 participates directly in cell binding and, if so, its mode of action, we conjugated purified gp80 to Covaspheres (Covalent Technology Corp., Ann Arbor, MI) and investigated their ability to bind to cells. The binding of gp80- Covaspheres was dependent on the developmental stage of the cells, with maximal interaction at the late aggregation stage. Scanning electron microscopic studies revealed the clustering of gp80-Covaspheres at the polar ends of these cells, similar to the pattern of gp80 distribution on the cell surface as reported earlier (Choi, A. H. C., and Siu, C.- H., 1987, J. Cell Biol., 104:1375-1387). Precoating cells with an adhesion-blocking anti-gp80 monoclonal antibody inhibited the binding of gp80-Covaspheres, suggesting that Covasphere-associated gp80 might undergo homophilic interaction with gp80 on the cell surface. Quantitative binding of 125I-labeled gp80 to intact cells gave an estimate of 1.5 X 10(5) binding sites per cell at the aggregation stage. Binding of soluble gp80 to cells was blocked by precoating cells with the anti-gp80 monoclonal antibody. The ability of gp80 to undergo homophilic interaction was further tested in a filter-binding assay, which showed that 125I-labeled gp80 was able to interact with gp80 bound on nitrocellulose in a dosage-dependent manner. In addition, reassociation of cells was significantly inhibited in the presence of soluble gp80, suggesting that gp80 has a single cell-binding site. These results are consistent with the notion that gp80 mediates cell- cell binding at the aggregation stage of development via homophilic interaction.