Involvement of Cell Surface Carbohydrates in the Sexual Cell Fusion of Dictyostelium discoideum

In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum ), monoclonal antibodies were raised against partially-purified gp 70, a fusion-related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion-blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N-linked glycosylation of proteins in cell fusion. All above results suggest that N-linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum .     

Cell Differentiation in Dictyostelium discoideum

We have shown previously that amoebae of D. discoideum strain V12 M2 starved at low density in the presence of cyclic AMP fail to form either stalk cells or prespore cells; a low molecular weight factor released by cells at high density promotes stalk formation under these conditions but formation of prespore cells requires 'cell contact'. Here we summarise evidence that:
1. Elevated intracellular cyclic AMP levels are required for all developmental gene expression beyond the preaggregative phase, and ammonia antagonises this expression in some way. However, the action of ammonia is not pathway specific.
2.'Cell contact' is a specific requirement for entry into the prespore pathway of gene expression since isolated cells provided with cyclic AMP synthesise much reduced amounts of the presporespecific enzyme uridine diphosphate (UDP) galactose polysaccharide transferase but normal amounts of the pathway-indifferent enzyme glycogen phosphorylase.
3. The 'cell contact' mechanism is uniquely sensitive to low concentrations of pronase. This protease selectively inhibits transferase synthesis and blocks in vitro spore differentiation (in a spore-forming mutant). It does not prevent chemotactic aggregation, stream formation, or stalk cell formation in the presence of cyclic AMP.     

Cell Fusion and Its Application to Genetic Analysis of Development in Dictyostelium discoideum

Dictyostelium cells were fused by a modification of the polyethylene glycol method of Kuhn and Parish. In the modified method Tricine buffer and Concanavalin A were used in place of Ca⁺⁺. The efficiency of genetic complementation through cell fusion was about 10 times higer by the modified method than by the original method with glycine buffer and Ca⁺⁺. Complementation between developmental mutants without any selectable growth character was clearly detected by the modified system, at efficiencies of about 1 in 10–20 surviving cells.     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.     

Cell Volume Determinations of Dictyostelium discoideum

The intercellular water present in pellets of centrifuged cell suspensions of the slime mold Dictyostelium discoideum was measured at four stages of differentiation.     

Cell Fusion Promoting Factor Common to Homothallic and Heterothallic Mating Systems in Dictyostelium discoideum1

Cellular slime mould Dictyostelium discoideum propagates as single haploid cells and under certain environmental conditions enters into a sexual cycle called macrocyst formation. There are homothallic and heterothallic strains reported, the former being able to form macrocysts in clonal cell populations while the latter to do so only in the presence of opposite mating-type strains. Molecular basis for differential mating systems is an intersting subject totally unknown yet. In the present study, sexual cell interactions in AC4, a homothallic strain of D. discoideum, was studied in comparison with the heterothallic mating system. The conditoned medium of AC4 cells was found to promote the sexual cell fusion among themselves. In addition, it also enhanced the cell fusion between heterothallic strains. Furthermore, the conditioned medium obtained from the mated culture of heterothallic strains reported to induce the sexual cell fusion in the heterothallic strains (Saga and Yanagisawa, 1983) was found also to promote the cell fusion in AC4. These results suggest that common regulatory mechanisms operate for sexual cell fusion among different mating systems in D. discoideum.     

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.     

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.     

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。     

Cell Migration: Regulation of cytoskeleton by Rap1 in Dictyostelium discoideum

Cell movement involves a coordinated regulation of the cytoskeleton, F-actin-mediated protrusions at the front and myosin-mediated contraction of the posterior of a cell. The small GTPase Rap1 functions as a key regulator in the spatial and temporal control of cytoskeleton reorganization for cell migration. This review outlines the establishment of cell polarity by differential localizations of the cytoskeleton and discusses the spatial and temporal regulation of cytoskeleton reorganization via the Rap1 signaling pathway during chemotaxis with a focus on recent advances in the study of chemotaxis using a simple eukaryotic model organism, Dictyostelium discoideum.     

Role of Cell Sorting in Pattern Formation in Dictyostelium discoideum

To examine the relationship between cell sorting and cell differentiation in the development of Dictyostelium discoideum , labeled cells grown in the absence of glucose [G(–) cells] and unlabeled cells grown in its presence [G(+) cells] were mixed and either allowed to undergo normal morphogenesis or cultured under submerged conditions. Changes in the distributions within a cell aggregate of labeled cells and cells stained with the conjugated antispore serum (prespore cells) were followed on the same sections by the methods of autoradiography and immunohistochemistry. In normal morphogenesis, differentiation of prespore cells apparently initiated and proceeded coincident with sorting out between G(+) and G(–) cells, during formation of a standing slug. By contrast, within an aggregate formed under submerged conditions, prespore cells began to differentiate long before G(+) and G(–) cells were sorted out, indicating that the cell sorting is not a prerequisite for the cell differentiation. The sorting out, however, brought about an accumulation of prespore cells in a hemisphere, thus producing a prestalk-prespore pattern within the aggregate.     

A Developmentally Regulated Kinesin-related Motor Protein from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive α-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end–directed microtubule motility in vitro at a speed of 0.14 μm/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.     

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans

The prestalk and prespore cells from the Dictyostelium discoideummulticellular slug stage of development differ in assembly ofglycoconjugates. Prespore cells are 2- to 3-fold more activethan prestalk cells in the assembly of N-linked glycans and20-fold more active in their fucosylation. Only prespore cellssynthesize an O-linked glycan consisting in part of Fuc -linkedto N-acetylglucosamine. Incorporation of fucose, glucosamine,mannose and galactose into large pronase-resistant glycoconjugateswas almost exclusively into prespore cells. Such glucosamine-labelledglycoconjugates resist fragmentation by ß-eliminationand include a glycoantigen dependent on the modB genetic locus.In contrast, large fucose-labelled glycoconjugates consistedof multiple, small, O-linked oligosaccharides on carrier peptides.The spore coat protein SP96 has several fucosylated O-linkedoligosaccharides, one of which correlates with a fucose epitopepreviously shown to localize in prespore vesicles and the outerlayer of the spore coat. Dictyostelium discoideum glycoconjugates glycoproteins prespore prestalk     

Spore Differentiation by Isolated Dictyostelium discoideum Cells, Triggered by Prior Cell Contact

Cells of D. discoideum mutant Fr-17 were allowed to form multicellular aggregates and develop undisturbed through 12 h (out of 18 required for terminal morphogenesis and cytodifferentiation). Then the cells were disaggregated and redeposited at densities so low as to preclude further sustained cell contacts and were incubated in the presence of certain diffusible metabolites. In this condition they transformed into spores and stalk cells with normal timing and, in the case of the spores, in proportions approaching those observed in undisturbed fruiting bodies. In contrast, mutant cells dispersed from aggregates at earlier stages or wild type cells dispersed from aggregates at any stage, remained as amoebae under the same conditions.
The completion of cytodifferentiation by the isolated cells was found to require threshold concentrations of diffusible, dialysable metabolites. A part of this requirement could be satisfied by addition of 10 mM NH₄Cl particularly in conjunction with an amino acid mixture. At least one metabolite, however, had to be supplied by feeder cells separated from the test cells by a dialysis membrane or by increasing the population density of the test cells themselves.     

Differential Cell Cohesiveness Expressed by Prespore and Prestalk Cells of Dictyostelium discoideum

Pseudoplasmodia of Dictyostelium discoideum at the culmination stage were separated into two cell populations by sedimentation in a discontinuous renografin gradient. The two lighter fractions (I and II) had enzymatic activities characteristic of the anterior prestalk cells, while the heaviest fraction (III) showed enzyme activities characteristic of the posterior prespore cells. Cell-cell adhesion among prespore cells is much more resistant to EDTA dissociation than 10-h cells and prestalk cells. Fab fragments prepared from antibodies directed against a specific cell surface glycoprotein gp150 were more effective in dissociating prespore cells than prestalk cells. In addition, prespore cells contained an approximately 2-fold higher concentration of the endogenous carbohydrate binding protein discoidin-I than prestalk cells. These differences may account for the differential cohesiveness of these two cell populations and provide a basis for cell recognition and cell sorting at the slug stage.     

Cell sorting within the prestalk zone of Dictyostelium discoideum

Abstract. The prestalk zone of slugs of Dictyostelium discoideum has been shown to contain three subregions in which the extracellular matrix genes ecmA and ecmB are differentially expressed; it is generally thought that these regions are defined by extracellular signals. Using β-galactosidase as a cell marker, we have shown that cells can sort specifically to all three regions. Cells from the posterior-prestalk zone ("prestalk 0 zone") which are injected into the slug tip move within 60 min back to their position of origin. When cells from the anterior prestalk zone (presumably containing a mixture of ecmA and ecmB expressers) are transplanted to the posterior prestalk zone, they move to the tip ("prestalk A zone") within 1 h and about 30 min subsequently are often found in a cone-shaped region within the tip ("prestalk B zone"). Cells transplanted to their own positions do not move significantly within this period. Since the sub-regions of the prestalk zone can be defined by sorting, it is possible that they are normally formed in this way rather than by position-dependent signals. Cells transplanted without a change in anterior-posterior position and cells which have sorted back to their positions of origin eventually spread out throughout the prestalk zone. This suggests that sorting preferences of cells are respecified. When posterior prestalk cells are transplanted to the prespore zone, respecification of sorting preference is suspended until the cells return to the prestalk zone and anterior-prestalk cells acquire posterior-prestalk sorting preferences.     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

Multidirectional phototaxis by Dictyostelium discoideum amoebae

Abstract Phototaxis by solitary Dictyostelium discoideum amoebae is known to be complex, the amoebae turning either towards or away from the light, depending on conditions such as light intensity. Having previously shown that amoebal phototaxis can be bidirectional (2 preferred directions either side of the light source), we now report the discovery of multidirectional phototaxis by D. discoideum amoebae, with up to 12 different preferred directions. As in the bidirectional case, multidirectional phototaxis depends on direction-dependent transitions from turning away from to turning towards the light source.